chapter 1 chapter 19 fluorophores and indicators for … · chapter 19 indicators for ca2+, mg2+,...

CHAPTER 19

Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions

Molecular Probes HandbookA Guide to Fluorescent Probes and Labeling Technologies

11th Edition (2010)

CHAPTER 1

Fluorophores and Their Amine-Reactive Derivatives

The Molecular Probes HandbookA GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES11th Edition (2010)

Molecular Probes Resources

Molecular Probes Handbook (online version)Comprehensive guide to uorescent probes and labeling technologies

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Fluorescence SpectraViewerIdentify compatible sets of uorescent dyes and cell structure probes

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BioProbes Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applications

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Access all Molecular Probes educational resources at lifetechnologies.com/mpeducate

Molecular Probes ResourcesMolecular Probes Handbook (online version)Comprehensive guide to fl uorescent probes and labeling technologiesthermofi sher.com/handbook

Molecular Probes Fluorescence SpectraViewerIdentify compatible sets of fl uorescent dyes and cell structure probesthermofi sher.com/spectraviewer

BioProbes Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applicationsthermofi sher.com/bioprobes

Access all Molecular Probes educational resources at thermofi sher.com/probes

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The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

NIN

ETEE

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CHAPTER 19

Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions

19.1 Introduction to Ca2+ Measurements with Fluorescent Indicators . . . . . . . . . . . . . . . . . . . . . . 833Selection Criteria for Fluorescent Ca2+ Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833

19.2 Fluorescent Ca2+ Indicators Excited with UV Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837Fura-2, Indo-1 and Related Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837

Fura-2 and Indo-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837

Fura-2 Calcium Imaging Calibration Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838

Bis-Fura-2: Brighter Signal with Lower Anity for Ca2+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838

Quin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838

Indicators with Intermediate Calcium-Binding Anity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839

Fura-4F and Fura-6F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839

Fura-FF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839

Low-Anity Calcium Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839

BTC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839

Mag-Fura-2 and Mag-Indo-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840

Data Table 19.2 Fluorescent Ca2+ Indicators Excited with UV Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840

Product List 19.2 Fluorescent Ca2+ Indicators Excited with UV Light. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 841

19.3 Fluorescent Ca2+ Indicators Excited with Visible Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842Fluo-3, Fluo-4, Rhod-2 and Related Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842

Fluo-3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842

Fluo-4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843

Rhod-2 and X-Rhod-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845

Rhod-3 Imaging Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 846

Lower-Anity Calcium Indicators Based on Fluo-3 and Rhod-2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 846

Fluo-5F, Fluo-4FF, Fluo-5N and Mag-Fluo-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 846

Rhod-5N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 847

Rhod-FF and X-Rhod-5F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 847

Indicators of Mitochondrial Ca2+ Transients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 847

Calcium Green, Calcium Orange and Calcium Crimson Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848

Calcium Green-1 and Calcium Green-2 Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848

Calcium Orange and Calcium Crimson Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 849

Calcium Green-5N and Magnesium Green: Low-Anity Ca2+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 849

Oregon Green 488 BAPTA Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 850

Oregon Green 488 BAPTA-1 and Oregon Green 488 BAPTA-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 850

Oregon Green 488 BAPTA-6F and Oregon Green 488 BAPTA-5N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851

Fura Red Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851

Calcein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851

Data Table 19.3 Fluorescent Ca2+ Indicators Excited with Visible Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 852

Product List 19.3 Fluorescent Ca2+ Indicators Excited with Visible Light. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853

The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies

IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

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Chapter 19 Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions

19.4 Fluorescent Ca2+ Indicator Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854Fura Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855

Visible-Excitation Ca2+ Indicator Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855

Data Table 19.4 Fluorescent Ca2+ Indicator Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857

Product List 19.4 Fluorescent Ca2+ Indicator Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857

19.5 Protein-Based Ca2+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 858Premo Cameleon Calcium Sensor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 858

Aequorin: A Bioluminescent Calcium Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 859

Properties and Applications of Aequorin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 859

Recombinant Aequorin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 860

Coelenterazine and Its Synthetic Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 860

Data Table 19.5 Protein-Based Ca2+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 861

Product List 19.5 Protein-Based Ca2+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 861

19.6 Fluorescent Mg2+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 862Magnesium Indicators Excited by UV Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 862

Mag-Fura-2 and Mag-Indo-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 862

Magnesium Indicators Excited by Visible Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 863

Magnesium Green Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 863

Mag-Fluo-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 863

Data Table 19.6 Fluorescent Mg2+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 864

Product List 19.6 Fluorescent Mg2+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 864

19.7 Fluorescent Indicators for Zn2+ and Other Metal Ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 865Applications of Ca2+ and Mg2+ Indicators for Detection of Zn2+ and Other Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 868

Indicators for Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869

FluoZin Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869

RhodZin-3 Indicator. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870

Newport Green DCF and Newport Green PDX Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870

TSQ. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870

Traditional Ca2+ and Mg2+ Indicators as Zn2+ Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870

Indicators for Copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871

Phen Green FL for Cu2+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871

Phen Green SK for Cu2+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871

Traditional Ca2+ and Mg2+ Indicators as Cu2+ Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871

Indicators for Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871

Phen Green FL and Phen Green SK Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871

Calcein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872

Indicators for Lead, Cadmium and Mercury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872

Leadmium Green Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872

Measure-iT Lead and Cadmium Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872

Phen Green FL and Phen Green SK Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872

Traditional Ca2+ and Mg2+ Indicators as Pb2+, Cd2+ and Hg2+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 873


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes




Indicators for Nickel and Cobalt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 873

Newport Green Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 873

Traditional Ca2+ and Mg2+ Indicators as Ni2+ and Co2+ Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 873

Indicators for Aluminum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 873

Indicators for Lanthanides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 873

Data Table 19.7 Fluorescent Indicators for Zn2+ and Other Metal Ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 874

Product List 19.7 Fluorescent Indicators for Zn2+ and Other Metal Ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 875

19.8 Chelators, Calibration Buers, Ionophores and Cell-Loading Reagents . . . . . . . . . . . . . . . . . 876Caged Calcium and Caged Calcium Chelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876

Nitrophenyl EGTA: A Superior Caged Calcium Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876

DMNP-EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876

Diazo-2: A Photoactivatable Calcium Knockdown Reagent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877

Nonuorescent Chelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877

BAPTA and BAPTA AM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877

Other BAPTA Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877

EGTA AM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878

DTPA Isothiocyanate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878

Calcium Sponge Polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878

TPEN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878

Calcium Calibration Buer Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878

Calcium Calibration Buer Kit #1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878

Fura-2 Calcium Imaging Calibration Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 879

Inux Pinocytic Cell-Loading Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 879

Loading P2X ReceptorExpressing Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 880

Ionophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 880

A-23187 and 4-Bromo A-23187 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 880

Ionomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 880

Probenecid: Inhibitor of Organic-Anion Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 880

Pluronic F-127 and PowerLoad Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 881

Pluronic F-127 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 881

PowerLoad Concentrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 881

Data Table 19.8 Chelators, Calibration Buers, Ionophores and Cell-Loading Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 881

Product List 19.8 Chelators, Calibration Buers, Ionophores and Cell-Loading Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 882



thermofisher.com/probes




Alexa Fluor 568 phalloidin, Oregon Green wheat germ agglutinin and Hoechst 33342.






Section 19.1 Introduction to Ca2+ Measurements with Fluorescent Indicators

19.1 Introduction to Ca2+ Measurements with Fluorescent IndicatorsFluorescent probes that show a spectral response upon binding

Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations using uorescence microscopy, ow cytom-etry and uorescence spectroscopy. e properties and applications of these uorescent indicatorsmost of which are derivatives of the Ca2+ chelators EGTA, APTRA and BAPTA 1have been extensively reviewed.24 Several earlier reviews of these ion indicators also contain useful technical information.58

We discuss chemical Ca2+ indicators according to their excitation requirements in Section 19.2 and Section 19.3, and their high-molecular weight conjugates are described in Section 19.4. Protein-based Ca2+ sen-sors are discussed in Section 19.5.

Selection Criteria for Fluorescent Ca2+ Indicators

We oer a wide selection of uorescent indicators available for detecting changes in intracellular Ca2+ over the range of 50 M (Table 19.1). As the primary suppliers of fura-2, indo-1, uo-3 (Figure 19.1.1), uo-4 and rhod-2, we also oer many specialized indicators for intracellular Ca2+. Our fura-4F, fura-6F and fura-FF indicators are designed to provide increased response sensitivity to intracellular Ca2+ concentration in the 0.55 M range, as compared with fura-2. e uo-3, uo-4, Oregon Green 488 BAPTA, Calcium Green, X-rhod-1 and Fura Red indicators and their variants enable Ca2+ detection in confocal microscopy and high-throughput G pro-teincoupled receptor (GPCR) screening applications. In addition, we oer indicators that are conjugated to high or lowmolecular weight dextrans for improved cellular retention and less compartmentaliza-tion (Section 19.4). We strive to provide the highest-purity indicators available anywhere. e AM ester forms of most of our indicators are typically at least 95% pure by HPLC analysis, although purity oen

Figure 19.1.1 A pyramidal neuron from rat hippocampus was rst exposed to Alzheimers -amyloid peptide and then to the excitatory amino acid glutamate. Confocal laser-scanning mi-croscopy imaging using the intracellular Ca2+ indicator uo-3 (F1241, F1242, F14218, F14242, F23915) shows that -amyloid peptide destabilizes the neurons calcium homeostasis and increases its vulnerability to excitotoxicity. The image was contributed by Mark P. Mattson, Sanders-Brown Center on Aging, University of Kentucky.

exceeds 98%. Furthermore, the AM esters of many of the Ca2+ and Mg2+ indicators are available in sets of 50 g for more convenient handling and reduced risk of deterioration during storage. For high-throughput screening applications, uo-3 AM and uo-4 AM are oered in special multi-unit packages (F14242, F14202), as well as in application-specic Fluo-4 NW and Fluo-4 Direct Calcium Assay Kits (Section 19.3).

A number of factors should be considered when selecting a uores-cent Ca2+ indicator, some of which are summarized in Table 19.1 and include the following:

Indicator form (salt, AM ester or dextran conjugate), which in-uences the cell-loading method and aects the indicators in-tracellular distribution and retention (Loading and Calibration of Intracellular Ion IndicatorsNote 19.1). e salt and dextran forms are typically loaded by microinjection, microprojectile bom-bardment or electroporation or by using the Inux pinocytic cell-loading reagent (I14402, Section 19.8) (Table 14.1). In contrast, the cell-permeant acetoxymethyl (AM) esters can be passively loaded into cells, where they are cleaved to cell-impermeant products by intracellular esterases.

Measurement mode, which is dictated by whether qualitative or quantitative ion concentration data are required. Ion indicators that exhibit spectral shis upon ion binding can be used for ratio-metric measurements of Ca2+ concentration, which are essentially independent of uneven dye loading, cell thickness, photobleaching eects and dye leakage (Loading and Calibration of Intracellular Ion IndicatorsNote 19.1). Excitation and emission wavelength preferences depend on the type of instrumentation being used, as well as on sample autouorescence and on the presence of other uorescent or photoactivatable probes in the experiment.

Dissociation constant (Kd), which must be compatible with the Ca2+ concentration range of interest. Indicators have a detectable response in the concentration range from approximately 0.1 Kd to 10 Kd. For ratiometric indicators, the Ca2+ response range is also somewhat dependent on the measurement wavelengths used.911 e Kd of Ca2+ indicators is dependent on many factors, including pH, temperature,1215 ionic strength, viscosity, protein binding and the presence of Mg2+ and other ions. Consequently, Kd values for intracellular indicators are usually signicantly higher than cor-responding values measured in cell-free solutions (Table 19.2).

Intracellular calibration of Ca2+ indicators may be achieved ei-ther by manipulating Ca2+ levels inside cells using an ionophore or by releasing the indicator into the surrounding medium of known Ca2+ concentration via detergent lysis of the cells. We also oer several com-pounds and buers for measuring and manipulating intracellular and extracellular Ca2+. ese products, which are discussed in Section 19.8, include caged Ca2+ reagents and caged chelators (NP-EGTA, DMNP-EDTA and diazo-2), as well as Calcium Calibration Buer Kits, BAPTA-derived buers, ion-selective chelating polymers (Calcium Sponge) and the important Ca2+ ionophores ionomycin, A-23187 and its nonuorescent analog, 4-bromo A-23187. Reagents for probing Ca2+ regulation and second-messenger activity are described in more detail in Chapter 17. Our reagents for the study of Ca2+ channels are described in Section 16.3.








Table 19.1 Summary of Molecular Probes uorescent Ca2+ indicators.

Ca2+ Indicator Water-Soluble Salt* Cell-Permeant Ester Dextran Mode Kd (nM)** Notes

Bis-fura-2t B6810 Ex340/380 370 1

BTC B6790 B6791 Ex400/480 7000 2

Calcium Green-1 C3010MP C3011MP, C3012 C6765, C3713, C3714 Em530 190 3, 4

Calcium Green-2 C3730 C3732 Em535 550 3, 5

Calcium Green-5N C3737 C3739 Em530 14,000 3

Calcium Orange C3013 C3015 Em575 185 2

Calcium Crimson C3018 Em615 185 2

Fluo-3 F1240, F3715 F1241, F1242, F14218, F14242, F23915 Em525 390 3, 4

Fluo-4 F14200 F14201, F14202, F14217, F23917 F14240, F36250 Em520 345 3, 6

Fluo-5F F14221 F14222 Em520 2300 3

Fluo-4FF F23980 F23981 Em520 9700 3

Fluo-5N F14203 F14204 Em520 90,000 3

Fura-2 F1200, F6799 F1201, F1221, F1225, F14185 F3029 Ex340/380 145 2

Fura-4F F14174 F14175 Ex340/380 770 2

Fura-6F F14178 Ex340/380 5300 2

Fura-FF F14180 F14181 Ex340/380 5500 2

Fura Red F14219 F3020, F3021 Ex420/480 140 2, 7

Indo-1 I1202 I1203, I1223, I1226 Em405/485 230 2

Mag-uo-4 M14205 M14206 Em520 22,000 3

Mag-fura-2 M1290 M1291, M1292 Ex340/380 25,000 2

Mag-indo-1 M1295 Em405/485 35,000 2, 8

Magnesium Green M3733 M3735 Em530 6000 3

Oregon Green 488 BAPTA-1 O6806 O6807 O6798 Em520 170 3

Oregon Green 488 BAPTA-2 O6808 O6809 Em520 580 3, 9

Oregon Green 488 BAPTA-6F O23990 Em520 3000 3

Oregon Green 488 BAPTA-5N O6812 Em520 20,000 3

Quin-2 Q23918 Em495 60 2, 10

Rhod-2 R14220 R1244, R1245MP R34676 Em580 570 3, 11

Rhod-3 R10145 Em580 570 3

Rhod-FF R23983 Em580 19,000 3

Rhod-5N R14207 Em580 320,000 3

X-rhod-1 X14210 Em600 700 3

X-rhod-5F X23984 X23985 Em600 1600 3*Catalog number(s) for the cell-impermeant salt. Catalog number(s) for the cell-permeant AM ester. Catalog number(s) for the dextran conjugates. Measurement wavelengths, in nm, where Ex = uorescence excitation and Em = uorescence emission. Indicators for which a pair of wavelengths are listed have dual-wavelength, ratio-measurement capability. **Ca2+ dissociation constant, measured in vitro at 22C in 100 mM KCl, 10 mM MOPS, pH 7.2, unless otherwise noted. Kd values depend on temperature, ionic strength, pH and other factors, and are usually higher in situ. Because indicator dextrans are intrinsically polydisperse and have variable degrees of substitution, these values may vary; lot-specic Kd values are printed on the vial in most cases. Low-anity dextran conjugate. High-anity dextran conjugate.Notes: 1. Ca2+-dependent uorescence response similar to fura-2 but ~75% greater molar absorptivity. 2. The AM ester form is uorescent (a major potential source of error in Ca2+ measurements). 3. The AM ester form is nonuorescent. 4. Calcium Green-1 is more uorescent than uo-3 in both Ca2+-bound and Ca2+-free forms. The magnitude of the Ca2+-dependent uorescence increase is greater for uo-3; see Section 19.3. 5. Larger Ca2+-dependent uorescence increase than Calcium Green-1. 6. The Kd value for the low-anity uo-4 dextran (F14240) is ~3 M, which is much higher than that of the free dye. The Kd value for the high-anity uo-4 dextran (F36250) is ~600 nM. 7. Can also be used in combination with uo-3 for dual-wavelength ratio measurements, Ex = 488 nm, Em = 530/670 nm (Cell Calcium (1995) 18:377; Cytometry (1994) 17:135; Cell Calcium (1993) 14:359). 8. Kd determined in 100 mM KCl, 40 mM HEPES, pH 7.0 at 22C (Biochem Biophys Res Commun (1991) 177:184). 9. Larger Ca2+-dependent uorescence increase than Oregon Green 488 BAPTA-1. 10. Kd determined in 120 mM KCl, 20 mM NaCl, pH 7.05 at 37C (Methods Enzymol (1989) 172:230). 11. The Kd value for the high-anity rhod dextran (R34676) is ~780 nM.

Table 19.2 Comparison of in vitro and in situ Kd values for various Ca2+ indicators.

Indicator Kd in vitro (nM)* Kd in situ (nM) Cell/Tissue Type

Calcium Green-1 190 930 HeLa cells 1

Fluo-3 390 2570 Frog skeletal muscle 2

Fluo-4 345 1000 HeLa cells 1

Fura-2 145 371 U373-MG astrocytoma cell 3

Fura-2 145 350 Rabbit gastric gland 4

Indo-1 230 844 Rabbit cardiac myocyte 5

Oregon Green 488 BAPTA-1 170 430 HeLa cells 1

Rhod-2 570 720 Mouse heart 6

* Values determined at 22C in 100 mM KCl, 10 mM MOPS, pH 7.2, 010 mM CaEGTA. Values determined in the cellular environments listed in the adjacent column. 1. Cell Calcium (2000) 28:213; 2. Biophys J (1993) 65:865; 3. Cell Calcium (1997) 21:233; 4. Methods Enzymol (1990) 192:38; 5. Biophys J (1995) 68:1453; 6. Cell Calcium (2001) 29:217.

REFERENCES1. Biochemistry (1980) 19:2396; 2. Methods Cell Biol (2007) 81:415; 3. Trends Mol Med (2008) 14:389; 4. Methods (2008) 46:143; 5. Physiol Rev (1999) 79:1089; 6. Methods Cell Biol (1994) 40:155; 7. Methods Enzymol (1990) 192:38; 8. Methods Enzymol (1989) 172:230; 9. Cell Calcium (1998) 24:17; 10. Methods Cell Biol (1994) 41:149; 11. Cell Calcium (1991) 12:29; 12. J Physiol (2002) 542:843; 13. Biophys J (2000) 78:2116; 14. Anal Biochem (1999) 273:60; 15. Biochem Biophys Res Commun (1990) 171:102.







There are two major prerequisites for measuring intracellular ion concentrations using uorescent indicators: Loading: The indicator must be localized in the region

(most commonly the cytosol but sometimes the mitochon-dria) where the ion concentration is to be measured.

Calibration: The uorescence of the indicator must be quantitatively related to the concentration of the free ion.

LoadingCell loading methods can be divided into two groups. Bulk loading

procedures are applicable to large populations of cells and include: Acetoxymethyl (AM) ester loading 47

ATP-induced permeabilization 8

Electroporation 911

Hypoosmotic shock 12

Inux pinocytic cell-loading reagent 13 (I14402, Section 19.8) Coupling to cell-penetrating peptides (CPP) 1416

Ballistic microprojectile delivery 1720

Procedures such as microinjection 21 and infusion from whole-cell patch pipettes 22 must be carried out one cell at a time. Reviews of some of these techniques have been published;23 see also Table 14.1.

The AM Ester Loading TechniqueThe noninvasive and technically straightforward AM ester technique

is by far the most popular method for loading uorescent ion indicators (Figure 1). The carboxylate groups of indicators for Ca2+ and other cat-ions and the phenolic hydroxyl groups of pH indicators are derivatized

NOTE 19.1

Loading and Calibration of Intracellular Ion Indicators

Figure 1 Schematic diagram of the processes involved in loading cells using membrane-permeant acetoxymethyl (AM) ester derivatives of uorescent indicators, in this case fura-2. Note the generation of potentially toxic by-products (formaldehyde and acetic acid).

as acetoxymethyl or acetate esters, respectively, rendering the indicator permeant to membranes and insensitive to ions. Once inside the cell, these derivatized indicators are hydrolyzed by ubiquitous intracellular esterases, releasing the ion-sensitive polyanionic indicator.

In practice, a 110 mM stock solution of the ester probe in anhydrous dimethylsulfoxide (DMSO) is prepared and divided into appropriately sized aliquots that can be stored desiccated at 20C. This procedure will curtail the spontaneous ester hydrolysis that can occur in moist environ-ments. Before loading, the DMSO stock solution should be diluted at least 1:200 in serum-free culture medium to a nal concentration of about 110 M. The nonionic and nondenaturing detergent Pluronic F-127 or the related PowerLoad reagent (P3000MP, P6866, P6867, P10020; Section 19.8) are frequently added to help disperse the indicator in the loading medium.24 After incubation at 2037C for 1560 minutes, the cells should be washed two to three times with fresh serum-free culture medium (serum may contain esterase activity). The loading medium should also be free of amino acids or buers containing primary or secondary amines because aliphatic amines may cleave the AM esters and prevent loading. The overall loading eciency is typically 1040%, depending on the molecular structure of the indicator, the type of cells and the incubation conditions.

Problems with AM Ester LoadingCompartmentalization: For calibration purposes, it is usually as-

sumed that uorescent indicators are homogeneously distributed in the cytosol and equally responsive to variations of intracellular ion concen-tration. However, AM esters and their hydrolysis products are capable of accumulating in any membrane-enclosed structure within the cell. In addition, indicators in polyanionic form may be sequestered within or-ganelles via active transport processes.25 Compartmentalization is usually more pronounced at higher loading temperatures and is particularly acute in plant and fungal cells.26,27 The extent of compartmentalization can be assessed by image analysis, as well as uorometrically using membrane permeabilization reagents, such as Triton X-100.24

Incomplete AM ester hydrolysis: Residual unhydrolyzed AM esters may be present extracellularly due to incomplete removal by washing. Inside the cell, low levels of intracellular esterase activity, which can vary considerably from one cell type to another, may produce only partial AM ester hydro-lysis.2830 Because even partially hydrolyzed AM esters are Ca2+-insensitive, detection of their uorescence as part of the total signal leads to an under-estimation of the Ca2+ concentration.31,32 Fluorescence quenching by Mn2+, which only binds with high anity to completely de-esteried indicators, can be used to quantitate these eects. Note that although some indicators are uorescent in the AM ester form, others are not (Table 19.1).

Extracellular AM ester hydrolysis: High levels of extracellular es-terase activity can make AM ester loading ineective, particularly for in vivo applications.33

Leakage: Extrusion of anionic indicators from cells by organic ion transporters can be reduced by cooling the sample or by applying inhibitors such as probenecid (P36400, Section 19.8), sulnpyrazone and MK571.25,34 AM esters have been shown to be extruded by the P-glycoprotein multidrug transporter 35 (Section 15.6). Ratiometric measurements help to minimize the impact of indicator leakage on experimental data.36

continued on next page








CalibrationIon Dissociation Constants

The dissociation constant (Kd) is the key conversion parameter linking uorescence signals to ion concentrations, assuming that the indicator is operating as an equilibrium sensor. This conventional assumption requires that the concentration of the indicator is close to the Kd value. Because intracel-lular indicator concentrations can easily reach 10100 M, even if the externally applied concentration is only 110 M, this assumption is not always valid.37 For pH indicators, Kd is conventionally expressed as its negative log (pKa). The concentration range over which an indicator produces an observable response is approximately 0.1 Kd to 10 Kd. For ratiometric measurements, the response range also depends on wavelength-dependent parameters.38,39 For BAPTA-based Ca2+ indicators in particular, the Kd is very sensitive to a number of environmental factors, including temperature, pH, ionic strength and interactions of the indicator with proteins.4043 Examination of published data shows that values of Kd determined in situ within cells can be up to 5-fold higher than values determined in vitro3,40,4446 (Table 19.2), underscoring the importance of performing calibrations to determine the Kd directly in the system under study.

Calibration MethodologyCalibration procedures basically consist of recording uorescence signals corresponding to a

series of precisely manipulated ion concentrations. The resulting sigmoidal titration curve is either linearized by means of a Hill plot or analyzed directly by nonlinear regression to yield Kd. For in vitro calibrations of Ca2+ indicators, EGTA buering is widely used to produce dened Ca2+ concentrations that can be calculated from the Kd of the Ca2+-EGTA complex.24,47,48 This technique is used in the Calcium Calibration Buer Kits (Section 19.8). In situ calibrations of intracellular indicators generally utilize an ionophore to equilibrate the controlled external ion concentration with the ion concentration within the cell.4,49 Commonly used ionophores include:

A-23187 (A1493), 4-bromo A-23187 (B1494) or ionomycin (I24222) for Ca2+ and Mg2+ (Section 19.8) Nigericin (N1495; Section 20.2, Section 21.2) for H+ and Cl

Gramicidin (G6888, Section 21.1) for Na+

Valinomycin (V1644, Section 21.1) for K+

Ratiometric CalibrationIndicators that show an excitation or emission spectral shift upon ion binding can be calibrated

using a ratio of the uorescence intensities measured at two dierent wavelengths, resulting in the cancellation of artifactual variations in the uorescence signal that might otherwise be misinterpreted as changes in ion concentration (Figure 2). Note that background levels must be subtracted from the component uorescence intensities before calculation of the ratio. Examples of indicators exhibiting ion-dependent spectral shifts include the Ca2+ indicators fura-2 (Figure 3) and indo-1 (Section 19.2), and the pH indicators BCECF and SNARF-1 (Section 20.2). The ratio of two intensities with opposite ion-sensitive responses (for example, 340 nm/380 nm in Figure 3) gives the largest possible dynamic range of ratio signals for a particular indicator. Alternatively, the ratio of an ion-sensitive intensity to an ion-insensitive intensity (measured at a spectral isosbestic point, e.g., 360 nm in Figure 3) can be used (Figure 2). Ratiometric measurements reduce or eliminate variations of several determining fac-tors in the measured uorescence intensity, including indicator concentration, excitation path length, excitation intensity and detection eciency.50,51 Artifacts that are eliminated include photobleaching and leakage of the indicator, variable cell thickness, and nonuniform indicator distribution within cells (due to compartmentalization) or among populations of cells (due to loading ecacy variations).

Figure 2 Simulated data demonstrating the practical importance of ratiometric uorescence techniques. This gure represents an ion indicator that exhibits a uo-rescence intensity increase in response to ion bind-ing at wavelength 1 and a corresponding decrease at 3. Fluorescence measured at an isosbestic point (2) is independent of ion concentration. The intracellular in-dicator concentration diminishes rapidly due to photo-bleaching, leakage (assuming the extracellular indicator is not detectable) or some other process. The change of intracellular ion concentration due to a stimulus applied at the time indicated by the arrow is unambiguously identied by recording the uorescence intensity ratios 1/3 or 1/2.

Figure 3 Fluorescence excitation spectra of fura-2 (F1200, F6799) in solutions containing 039.8 M free Ca2+

39.8 M free Ca2+Em = 510 nm

Fluo

resc

ence

exc

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n

Wavelength (nm)250 300300 350 400 450

1.350.60

0.350.23

0.150.10

0.065

0.0380.017

0

1. Methods Cell Biol (2007) 81:415; 2. Methods (2008) 46:143; 3. Cell Calcium (2000) 28:213; 4. Methods Mol Biol (2006) 312:229; 5. Methods Mol Biol (2009) 489:93; 6. Nat Protoc (2006) 1:380; 7. Nature (1981) 290:527; 8. J Biol Chem (1987) 262:8884; 9. Nat Protoc (2009) 4:862; 10. Neuron (2007) 53:789; 11. J Neurophysiol (2005) 93:1793; 12. Cytometry (1997) 28:316; 13. Biotechniques (2002) 33:358; 14. Bioconjug Chem (2009) 20:249; 15. Org Biomol Chem (2008) 6:4516; 16. Nano Lett (2004) 4:2019; 17. Plant J (2006) 46:327; 18. J Neurosci Methods (2009) 184:332; 19. J Neurosci Methods (2005) 141:41; 20. Methods (2003) 30:79; 21. Biochem J (1994) 302:5; 22. CSH Protoc (2009) 2009:pdb.prot5201; 23. Proc Natl Acad Sci U S A (2001) 98:4295; 24. Methods Cell Biol (1994) 40:155; 25. Cell Calcium (1990) 11:57; 26. J Exp Biol (1994) 196:419; 27. J Microsc (1992) 166:57; 28. Cell Calcium (1990) 11:63; 29. Am J Physiol (1988) 255:C304; 30. Anal Biochem (1988) 169:159; 31. Biophys J (1994) 67:476; 32. Biophys J (1993) 65:561; 33. J Microsc (2007) 226:74; 34. J Neurosci Methods (2008) 167:140; 35. J Biol Chem (1993) 268:21493; 36. Anal Biochem (2009) 390:212; 37. Mol Pharmacol (2002) 62:618; 38. Cell Calcium (1991) 12:29; 39. Cell Calcium (1998) 24:17; 40. Biophys J (1994) 67:1646; 41. Biophys J (1992) 63:89; 42. Biochem Biophys Res Commun (1991) 180:209; 43. Biochem Biophys Res Commun (1991) 177:184; 44. Cell Calcium (2003) 34:1; 45. Biophys J (1995) 68:1453; 46. Biophys J (1993) 65:865; 47. Methods Enzymol (1989) 172:230; 48. Cell Calcium (1991) 12:279; 49. Cell Calcium (1997) 21:233; 50. Methods Cell Biol (1989) 30:157; 51. J Biol Chem (1985) 260:3440.

continued from previous page






Section 19.2 Fluorescent Ca2+ Indicators Excited with UV Light

19.2 Fluorescent Ca2+ Indicators Excited with UV Light

Figure 19.2.1 False-color image of free Ca2+ concentra-tion in a Purkinje neuron from embryonic mouse cerebel-lum. Neurons were grown in dispersed tissue culture for 12 days, loaded with the pentapotassium salt of fura-2 (F1200) using a microelectrode and then challenged with trans-ACPD, an agonist of metabotropic glutamate receptors, in the ab-sence of extracellular Ca2+. The composite image, which rep-resents the ratio of images obtained with excitation at 340 nm and 380 nm, reveals the mobilization of internal Ca2+ stores without contribution from Ca2+ inux. The image was contrib-uted by D.J. Linden, Johns Hopkins University, and M. Smeyne and J.A. Connor, Roche Institute of Molecular Biology.

Figure 19.2.2 Fluorescence excitation spectra of fura-2 (F1200, F6799) in solutions containing 039.8 M free Ca2+.


Fluo

resc

ence

exc

itatio

n

Wavelength (nm)250 300300 350 400 450

1.350.60

0.350.23

0.150.10

0.065

0.0380.017

0

Figure 19.2.3 Fluorescence emission spectra of indo-1 (I1202) in solutions containing 039.8 M free Ca2+.

39.8 M free Ca2+Ex = 338 nm

Fluo

resc

ence

em

issi

on

Wavelength (nm)350 400 450 500 550 600

1.35

0.60

0.350.230.15

0.100.065

0.0380.017

0

Fura-2, Indo-1 and Related DerivativesFura-2 and Indo-1

Fura-2 and indo-1 are UV lightexcitable, ratiometric Ca2+ indicators. Fura-2 has become the dye of choice for ratio-imaging microscopy (Figure 19.2.1), in which it is more practical to change excitation wavelengths than emission wavelengths.1,2 Upon binding Ca2+, fura-2 exhib-its an absorption shi that can be observed by scanning the excitation spectrum between 300 and 400 nm, while monitoring the emission at ~510 nm (Figure 19.2.2). In contrast, indo-1 is a preferred dye for ow cytometry, where it is more practical to use a single laser for excitationusually the 351364 nm spectral lines of the argon-ion laserand monitor two emissions.3,4 e emission maximum of indo-1 shis from ~475 nm in Ca2+-free medium to ~400 nm when the dye is saturated with Ca2+ (Figure 19.2.3).

Modern two-photon excitation imaging techniques used with fura-2 and indo-1 59 avoid the deleterious eects of conventional ultraviolet illumination on live specimens. Indo-1 may be less subject to compartmentalization than fura-2,10 whereas fura-2 is more resistant to pho-tobleaching than indo-1.11,12 Both fura-2 and indo-1 exhibit Kd values that are close to typical basal Ca2+ levels in mammalian cells (~100 nM) and display high selectivity for Ca2+ binding relative to Mg2+.13 Nevertheless, Ca2+ binding is discernibly perturbed by physiological levels of Mg2+; the Kd for Ca2+ of fura-2 is ~135 nM in Mg2+-free Ca2+ buers and ~224 nM in the presence of 1 mM Mg2+ (measured at 37C in 100 mM KCl, 10 mM MOPS, pH 7.0). 13 Fura-2 and indo-1 also exhibit high anities for other divalent cations such as Zn2+ and Mn2+, a property that is discussed further in Section 19.7.

e sodium and potassium salts of fura-2 (F6799, F1200; Figure 19.2.4; Figure 19.2.5) and potassium salt of indo-1 (I1202, Figure 19.2.6) are cell-impermeant probes that can be delivered into cells by microinjection or using our Inux pinocytic cell-loading reagent (I14402, Section 19.8). Free acids of fura-2 and indo-1 can also be loaded into some plant cells at pH 45.1418 In addition, these salts are useful as standards for calibrating Ca2+ measurements.

Figure 19.2.6 Indo-1, pentapotassium salt (I1202).Figure 19.2.4 Fura indicators with varying Ca2+ anities.

R6

R6

R4

R4

OCH2CH2O

(OCCH2)2N

O

O O

N(CH2CO )2

N

O

C

O

O

Indicator

0.14 M

0.40 M

0.77 M

5.30 M

5.50 M

H

H

F

H

H

CH3F

H

H

F

H

H

H

F

F

Fura-2

Fura-5F

Fura-4F

Fura-6F

Fura-FF

Kd(Ca2+)

R5

R5

Figure 19.2.5 Fura-2, pentapotassium salt (F1200).








Figure 19.2.7 Fura-2, AM (F1201).

Figure 19.2.8 Indo-1, AM (I1203).

Unlike the salt forms, the acetoxymethyl (AM) esters of fura-2 (Figure 19.2.7) and indo-1 (Figure 19.2.8) can passively diuse across cell membranes, enabling researchers to avoid the use of invasive loading techniques. Once inside the cell, these esters are cleaved by intra-cellular esterases to yield cell-impermeant uorescent indicators (Loading and Calibration of Intracellular Ion IndicatorsNote 19.1). We oer fura-2 AM and indo-1 AM in 1 mg vials (F1201, I1203) or specially packaged in 20 vials of 50 g each (F1221, I1223); the special pack-aging is recommended when small quantities of the dyes are to be used over a long period of time. We also provide stock solutions of fura-2 AM and indo-1 AM in anhydrous DMSO at 1 mg/mL (~1 mM; F1225, I1226). Our standard analytical specications for fura-2 AM require 95% purity by HPLC. We also oer a special packaged high-purity grade of fura-2 AM that is specied to have 98% purity by HPLC (as a set of 20 vials, each containing 50 g; F14185). e 10,000 MW dextran conjugate of fura is described in Section 19.4.

Fura-2 Calcium Imaging Calibration Kite Fura-2 Calcium Imaging Calibration Kit (F6774) is designed to facilitate rapid calibra-

tion and standardization of digital imaging microscopes.2,19 is kit provides 11 CaEGTA:EGTA buer solutions with free Ca2+ concentrations from zero (10 mM EGTA) to 39 M. Each solution also includes 50 M fura-2, as well as 15 m unstained polystyrene microspheres to act both as spacers that ensure uniform separation between the slide and the coverslip and as focusing aids. We also provide a twelh bueridentical to the 10 mM CaEGTA standard but lacking fura-2that serves as a control for background uorescence. Our Calcium Calibration Kits are described further in Section 19.8.

Bis-Fura-2: Brighter Signal with Lower Anity for Ca2+By linking two fura uorophores with one BAPTA chelator (Figure 19.2.9), we have pro-

duced bis-fura-2, a Ca2+ indicator that exhibits approximately twice the absorptivity of fura-2. Bis-fura-2 has a Kd for Ca2+ of ~370 nM and ~525 nM in the absence and presence of 1 mM Mg2+, respectively (measured at ~22C using our Calcium Calibration Buer Kits). In other aspects, the quantum yield of bis-fura-2 and its spectral response to Ca2+ (Figure 19.2.10) are virtually identical to those of fura-2. Although the dierence between the Kd of fura-2 and bis-fura-2 for Ca2+ is small, the change in excitation ratio for bis-fura-2 in response to Ca2+ concentrations >500 nM is larger than that of fura-2 (Figure 19.2.2); this dierence can im-prove the dynamic range for Ca2+ measurements in cells.20,21 Other potential advantages of bis-fura-2 include:

Higher uorescence output per indicator, which may allow the use of lower dye concentrations 21

Lower anity for Ca2+, which decreases the buering of intracellular Ca2+ and produces a faster response to Ca2+ spikes 20

An additional negative charge, which may facilitate dye retention

e hexapotassium salt of bis-fura-2 (B6810) is available for loading by microinjection 22,23 or by infusion from a patch pipette.24 We do not currently oer a membrane-permeant AM ester of bis-fura-2.

Quin-2Quin-2 belongs to the rst generation of Ca2+ indicators developed by Tsien 25 (Figure

19.2.11). Quin-2 has lower absorptivity and quantum yield values than the fura-2, indo-1, uo-3, uo-4 and Calcium Green indicators and thus requires higher loading concentrations. e resulting high intracellular concentration of the indicator may buer intracellular Ca2+ tran-sients.26 Quin-2 AM has been used to intentionally deplete cytosolic free Ca2+ 27,28 and to en-sure unidirectional Ca2+ inux.29 Measurement of cytosolic free Ca2+ with quin-2 has been thoroughly reviewed by Tsien and Pozzan.30 We oer quin-2 as a high-purity, cell-impermeant free acid (Q23918).

Figure 19.2.9 Bis-fura-2, hexapotassium salt (B6810).

Figure 19.2.11 Quin-2, free acid (Q23918).

Figure 19.2.10 Fluorescence excitation spectra of bis-fu-ra-2 (B6810) in solutions containing 039.8 M free Ca2+.


Fluo

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exc

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n

Wavelength (nm)250 300300 350 400 450

1.700.60

0.350.23

0.150.10

0.065

0.0380.0170







Indicators with Intermediate Calcium-Binding AnityFura-4F and Fura-6F

Calcium concentrations above 1 M produce almost complete binding saturation of fura-2 but very low fractional saturation of the low-anity fura analog mag-fura-2 (M1290). To bridge this gap in the Ca2+ measurement range of fura-type indicators, we currently of-fer two additional ratiometric Ca2+ indicatorsfura-4F (F14174) and fura-6F (F14178)as well as the membrane-permeant fura-4F AM 31 (F14175); fura-5F may be available upon request at www.invitrogen.com/handbook/customorganics. Attachment of a single electron-withdrawing uorine substituent at dierent positions on the BAPTA chelator moiety of fura-2 (Figure 19.2.4) results in an increase of the Kd value to ~770 nM, ~400 nM and 5.3 M for fura-4F, fura-5F and fura-6F, respectively (measured at 22C at 100 mM KCl, 10 mM MOPS, pH 7.2). Except for the change in the Ca2+ concentration response range (Figure 19.2.12), the Ca2+-dependent spectral shis produced by fu-ra-4F, fura-5F and fura-6F are essentially identical to those of fura-2 (Figure 19.2.13) and the probes use the same optical lter sets.

Fura-FFFura-FF is a diuorinated derivative of fura-2 (Figure 19.2.4)

with a Kd value of ~5.5 M 3234 (measured at 22C in 100 mM KCl, 10 mM MOPS, pH 7.2) and similar spectroscopic properties (Figure 19.2.14). Fura-FF has negligible Mg2+ sensitivity, making Ca2+ detec-tion less susceptible to interference than with mag-fura-2.3234 ese

Figure 19.2.12 Fluorescence excitation ratio versus Ca2+ concentration curves for fura-2 (red), fura-5F (orange), fura-4F (green) and fura-6F (blue).

-8.0

Exc

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tio (3

40/3

80 n

m)

log [Ca2+]

0

50

40

30

20

10

-7.0 -6.0 -5.0 -4.0 -3.0

Figure 19.2.13 Ca2+-dependent uorescence excitation spectra of fura-4F (F14174).

Wavelength (nm)300 350 400 450

Em = 510 nm

5.002.85

1.350.60

Fluo

resc

ence

exc

itatio

n 75.0 M free Ca2+

0.350.230.100

Figure 19.2.14 Ca2+-dependent uorescence excitation spectra of fura-FF (F14180).

Fluo

resc

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exc

itatio

n

Wavelength (nm)300 400 450

Em = 510 nm

30

350

1000 M free Ca2+

20105

2.85

1.35

0

Figure 19.2.15 BTC, tetrapotassium salt (B6790).

properties have made fura-FF particularly useful for spatial and func-tional characterization of intracellular Ca2+ stores 35 and for tracking Ca2+ oscillations driven by the inositol 1,4,5-triphosphate receptor.36,37 e low-anity indicator fura-FF detected NMDA- and kainate-in-duced neuronal Ca2+ uxes that were not detectable with the higher-anity indicator fura-2.38 Fura-FF has also been used in combination with FluoZin-3 (Section 19.7) for simultaneous detection of Ca2+ and Zn2+.39 Fura-FF is available in water-soluble potassium salt form (F14180) and as a membrane-permeant AM ester derivative (F14181).

Low-Anity Calcium IndicatorsBTC

e coumarin benzothiazolebased Ca2+ indicator BTC (B6790, Figure 19.2.15) and its cell-permeant derivative BTC AM (B6791) were developed in collaboration with Haralambos Katerinopoulos of the University of Crete.40 is Ca2+ indicator exhibits a shi in excitation maximum from about 480 nm to 400 nm upon binding Ca2+ (Figure 19.2.16), permitting ratiometric measurements that are essentially in-dependent of uneven dye loading, cell thickness, photobleaching and dye leakage.41,42 Its high selectivity and moderate anity for Ca2+ (Kd ~7 M) 42 allows accurate quantitation of high intracellular Ca2+ lev-els that are underestimated by fura-2 measurements.43,44 When loaded into neurons as its AM ester, BTC exhibits little compartmentalization; however, prolonged excitation appears to cause conversion of the indi-cator to a calcium-insensitive form.44

Figure 19.2.16 Fluorescence excitation spectra of BTC (B6790) in solutions containing 0100 M free Ca2+.

Fluo

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exc

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400 440

Wavelength (nm)480 520

1.3

Em = 540 nm

6

1020

50

100 M Ca2+

0








BTC has been employed in investigations of Ca2+-dependent exo-cytosis in pancreatic -cells,45 CHO broblasts 46 and phaeochromo-cytoma cells.47,48 Neuronal Ca2+ transients detected by the low-anity Ca2+ indicators BTC and mag-fura-2 are signicantly more rapid than those reported by the higher-anity indicators fura-2 and Calcium Green-2.44,49

Mag-Fura-2 and Mag-Indo-1Mag-fura-2 (also called furaptra, Figure 19.2.17) and mag-indo-1

were originally designed to report intracellular Mg2+ levels (Section 19.6); however, these indicators actually have much higher anity for Ca2+ than for Mg2+. Although Ca2+ binding by these indicators may complicate analysis when they are employed to measure intracellular Mg2+,50,51 their increased eective range and improved linearity for Ca2+ measurements has been exploited for measuring intracellular Ca2+ levels between 1 M and 100 M.35

e spectral shis of mag-fura-2 and mag-indo-1 are very simi-lar to those of fura-2 and indo-1 but occur at higher Ca2+ concentra-tions. Because the o-rates for Ca2+ binding of these indicators are much faster than those of fura-2 and indo-1, these dyes have been used to monitor action potentials in skeletal muscle and nerve terminals with little or no kinetic delay 5255 (Figure 19.2.18). e spectral proper-ties, kinetics and selectivity of several of our low-anity Ca2+ indica-tors have been reviewed by Zhao,56 Hyrc 32 and their co-workers.

e moderate Ca2+ anity of mag-fura-2 and the tendency of its AM ester to accumulate in subcellular compartments have proven use-ful for in situ monitoring of inositol 1,4,5-triphosphatesensitive Ca2+ stores.35,57 Mag-fura-2 has also been employed to follow Ca2+ transients in presynaptic nerve terminals,49,5860 gastric epithelial cells 61 and cul-tured myocytes.62 Mag-indo-1 has been used to detect gonadotropin-releasing hormoneinduced Ca2+ oscillations in gonadotropes 63 and to investigate the role of Ca2+/K+ exchange in intracellular Ca2+ storage and release processes.64 Mag-fura-2 (M1290, M1291, M1292) and mag-indo-1 (M1295) are available as cell-impermeant potassium salts or as cell-permeant AM esters.

Figure 19.2.18 Ca2+ transients evoked by trains of 14 action potentials in rat cerebellar gran-ule cells detected by fura-2 (upper panel, F1200) and mag-fura-2 (lower panel, M1290). The stimulus pulses are 50 milliseconds apart (20 Hz); timing is indicated by the double-headed ar-rows. The amplitude of the transients detected by fura-2 decreases with each successive stimu-lus due to Ca2+ saturation. Mag-fura-2 avoids saturation due to its lower Ca2+ binding anity (Kd for Ca2+ = 25 M), recording transients of approximately equal amplitude from successive action potentials. Adapted with permission from Biophys J (1995) 68:2165.

Rel

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REFERENCES1. Methods Cell Biol (2007) 81:415; 2. J Vis Exp (2009) doi: 10.3791/1067; 3. Methods Mol Biol (2010) 612:149; 4. Methods (2000) 21:221; 5. Nature (2009) 460:264; 6. J Neurophysiol (2007) 97:3118; 7. Nat Protoc (2006) 1:380; 8. Biophys J (2004) 86:1726; 9. Nat Neurosci (2000) 3:452; 10. Cell Calcium (1990) 11:487; 11. Chem Biol (1996) 3:765; 12. Am J Physiol (1987) 253:C613; 13. J Biol Chem (1985) 260:3440; 14. Proc Natl Acad Sci U S A (1992) 89:3591; 15. Plant Physiol (1990) 93:841; 16. Plant Sci (1990) 67:125; 17. Cell Calcium (1987) 8:455; 18. Eur J Cell Biol (1988) 46:466; 19. J Biol Chem (2005) 280:31936; 20. Pugers Arch (2008) 456:267; 21. Biophys J (1998) 75:1635; 22. J Mol Cell Cardiol (2010) 48:1023; 23. Am J Physiol Heart Circ Physiol (2007) 292:H2212; 24. J Neurosci (2006) 26:3482; 25. Biochemistry (1980) 19:2396; 26. J Biol Chem (1983) 258:4876; 27. J Biol Chem (1998) 273:8203; 28. Brain Res (1990) 528:48; 29. Biochemistry (1987) 26:6995; 30. Methods Enzymol (1989) 172:230; 31. Bioorg Med Chem Lett (2000) 10:1515; 32. Cell Calcium (2000) 27:75;

33. Biophys J (1999) 76:2029; 34. Am J Physiol (1994) 266:C1313; 35. Methods Mol Biol (2006) 312:229; 36. J Biol Chem (1999) 274:14157; 37. EMBO J (1997) 16:3533; 38. J Neurosci (1998) 18:7727; 39. Cell Calcium (2005) 37:225; 40. Cell Calcium (1994) 15:190; 41. Biochem Biophys Res Commun (2004) 317:77; 42. J Physiol (2001) 533:757; 43. J Neurosci (1997) 17:6669; 44. Cell Calcium (1998) 24:165; 45. J Cell Biol (1997) 138:55; 46. J Biol Chem (1996) 271:17751; 47. J Physiol (2001) 533:627; 48. J Physiol (1996) 494:53; 49. Biophys J (1995) 68:2156; 50. Anal Biochem (2001) 290:221; 51. Am J Physiol (1992) 263:C300; 52. Proc Natl Acad Sci U S A (1996) 93:8095; 53. J Physiol (1994) 475:319; 54. Biochem Biophys Res Commun (1991) 177:184; 55. J Gen Physiol (1991) 97:271; 56. Biophys J (1996) 70:896; 57. J Cell Sci (2006) 119:226; 58. J Physiol (2000) 527:33; 59. Biophys J (1997) 72:1458; 60. Biophys J (1997) 72:637; 61. Am J Physiol (1994) 267:G442; 62. Biophys J (2005) 88:1911; 63. Proc Natl Acad Sci U S A (1994) 91:9750; 64. Nature (1998) 395:908.

Figure 19.2.17 Mag-fura-2, tetrapotassium salt (M1290).

O

NO

N(CH2CO)2OCH2CO

O

O

COO

4 K

DATA TABLE 19.2 FLUORESCENT Ca2+ INDICATORS EXCITED WITH UV LIGHT Low Ca2+ High Ca2+

Cat. No. MW Storage Soluble Abs EC Em Solvent Abs EC Em Solvent Product Kd NotesB6790 844.03 F,D,L pH >6 464 29,000 533 H2O 401 20,000 529 H2O/Ca2+ 7.0 M 1, 2, 3B6791 979.92 F,D,L DMSO 433 39,000 504 MeOH B6790B6810 1007.14 F,D,L pH >6 366 56,000 511 H2O 338 68,000 504 H2O/Ca2+ 370 nM 1, 2, 4, 5







DATA TABLE 19.2 FLUORESCENT Ca2+ INDICATORS EXCITED WITH UV LIGHTcontinued Low Ca2+ High Ca2+

Cat. No. MW Storage Soluble Abs EC Em Solvent Abs EC Em Solvent Product Kd NotesF1200 832.00 F,D,L pH >6 363 28,000 512 H2O 335 34,000 505 H2O/Ca2+ 145 nM 1, 2, 4, 5F1201 1001.86 F,D,L DMSO 370 31,000 476 EtOAc F1200F1221 1001.86 F,D,L DMSO 370 31,000 476 EtOAc F1200F1225 1001.86 F,D,L DMSO 370 31,000 476 EtOAc F1200 6F6799 751.45 F,D,L pH >6 363 28,000 512 H2O 335 34,000 505 H2O/Ca2+ 145 nM 1, 2, 4, 5F14174 835.96 F,D,L pH >6 366 21,000 511 H2O 336 23,000 505 H2O/Ca2+ 770 nM 1, 2, 4F14175 1005.82 F,D,L DMSO 370 29,000 475 EtOAc F14174F14178 835.96 F,D,L pH >6 364 25,000 512 H2O 336 28,000 505 H2O/Ca2+ 5.3 M 1, 2, 3F14180 853.95 F,D,L pH >6 364 25,000 510 H2O 335 28,000 506 H2O/Ca2+ 5.5 M 1, 2, 3F14181 1023.82 F,D,L DMSO 370 30,000 476 EtOAc F14180F14185 1001.86 F,D,L DMSO 370 31,000 476 EtOAc F1200 7I1202 840.06 F,D,L pH >6 346 33,000 475 H2O 330 33,000 401 H2O/Ca2+ 230 nM 1, 2, 4, 5I1203 1009.93 F,D,L DMSO 356 39,000 478 MeOH I1202I1223 1009.93 F,D,L DMSO 356 39,000 478 MeOH I1202I1226 1009.93 F,D,L DMSO 356 39,000 478 MeOH I1202 6M1290 586.68 F,D,L pH >6 369 22,000 511 H2O 329 26,000 508 H2O/Ca2+ 25 M 1, 2, 3M1291 722.57 F,D,L DMSO 366 31,000 475 EtOAc M1290M1292 722.57 F,D,L DMSO 366 31,000 475 EtOAc M1290mag-indo-1 594.74 F,D,L pH >6 349 38,000 480 H2O 328 35,000 390 H2O/Ca2+ 35 M 1, 2, 8, 9M1295 730.63 F,D,L DMSO 354 37,000 472 MeOH mag-indo-1Q23918 541.51 D,L pH >6 353 4000 495 H2O 333 3900 495 H2O/Ca2+ 60 nM 1, 2, 10For denitions of the contents of this data table, see Using The Molecular Probes Handbook in the introductory pages.Notes

1. Dissociation constants are known to vary considerably depending on the temperature, pH, ionic strength, viscosity, protein binding, presence of other ions (especially polyvalent ions), instru-ment setup and other factors. It is strongly recommended that these values be veried under user-specic experimental conditions.

2. Spectra measured in aqueous buers containing 10 mM EGTA (H2O) or a >10-fold excess of free Ca2+ relative to the Kd (H2O/Ca2+).3. Dissociation constant determined by uorescence measurements in 100 mM KCl, 10 mM MOPS, pH 7.2, 0 to 1 mM free Ca2+ at 22C.4. Dissociation constant determined by uorescence measurements in 100 mM KCl, 10 mM MOPS, pH 7.2, 0 to 39 M free Ca2+ at 22C.5. Kd(Ca2+) for fura-2 and indo-1 from the original reference by Grynkiewicz, Poenie and Tsien (J Biol Chem (1985) 260:3440) are 224 nM and 250 nM, respectively, measured in 1 mM EGTA, 100 mM KCl,

1 mM free Mg2+, 10 mM MOPS, pH 7.0 at 37C. For bis-fura-2, Kd(Ca2+) in presence of Mg2+ is 525 nM (determined in our laboratories using 100 mM KCl, 10 mM MOPS, pH 7.2, 1 mM Mg2+ at 22C).6. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."7. This product is specied to equal or exceed 98% analytical purity by HPLC.8. The emission spectrum of Ca2+-bound mag-indo-1 excited at 340 nm has approximately equal peak intensities at ~390 nm and ~480 nm. (Biochemistry (1991) 30:702)9. Dissociation constant determined in 100 mM KCl, 40 mM HEPES, pH 7.0 at 22C. (Biochem Biophys Res Commun (1991) 177:184)10. Kd(Ca2+) for quin-2 was measured in 120 mM KCl, 20 mM NaCl, pH 7.05 at 37C. Under the same conditions with addition of 1 mM Mg2+, Kd = 115 nM. (Methods Enzymol (1989) 172:230)

PRODUCT LIST 19.2 FLUORESCENT Ca2+ INDICATORS EXCITED WITH UV LIGHTCat. No. Product QuantityB6810 bis-fura-2, hexapotassium salt *cell impermeant* 1 mgB6791 BTC, AM *cell permeant* 100 gB6790 BTC, tetrapotassium salt *cell impermeant* 1 mgF6774 Fura-2 Calcium Imaging Calibration Kit *zero to 10 mM CaEGTA, 50 M fura-2 (11 x 1 mL)* 1 kitF1225 fura-2, AM *1 mM solution in anhydrous DMSO* *cell permeant* 1 mLF1201 fura-2, AM *cell permeant* 1 mgF1221 fura-2, AM *cell permeant* *special packaging* 20 x 50 gF14185 fura-2, AM *FluoroPure grade* *special packaging* 20 x 50 gF1200 fura-2, pentapotassium salt *cell impermeant* 1 mgF6799 fura-2, pentasodium salt *cell impermeant* 1 mgF14175 fura-4F, AM *cell permeant* *special packaging* 10 x 50 gF14174 fura-4F, pentapotassium salt *cell impermeant* 500 gF14178 fura-6F, pentapotassium salt *cell impermeant* 500 gF14181 fura-FF, AM *cell permeant* *special packaging* 10 x 50 gF14180 fura-FF, pentapotassium salt *cell impermeant* 500 gI1226 indo-1, AM *1 mM solution in anhydrous DMSO* *cell permeant* 1 mLI1203 indo-1, AM *cell permeant* 1 mgI1223 indo-1, AM *cell permeant* *special packaging* 20 x 50 gI1202 indo-1, pentapotassium salt *cell impermeant* 1 mgM1291 mag-fura-2, AM *cell permeant* 1 mgM1292 mag-fura-2, AM *cell permeant* *special packaging* 20 x 50 gM1290 mag-fura-2, tetrapotassium salt *cell impermeant* 1 mgM1295 mag-indo-1, AM *cell permeant* *special packaging* 20 x 50 gQ23918 quin-2, free acid *cell impermeant* 5 mg







Section 19.3 Fluorescent Ca2+ Indicators Excited with Visible Light

19.3 Fluorescent Ca2+ Indicators Excited with Visible LightFluo-3, Fluo-4, Rhod-2 and Related DerivativesFluo-3

e Ca2+ indicator uo-3 (Figure 19.3.1) was developed by Tsien and colleagues for use with visible-light excitation sources in ow cytometry and confocal laser-scanning microscopy.1 More recently, imaging with uo-3 and its derivatives has been extended to include two-photon excitation techniques 25 (Figure 19.3.2) and total internal reection uorescence (TIRF) microscopy.6 Fluo-3 imaging has revealed the spatial dynamics of many elementary processes in Ca2+ signaling 711 (Figure 19.3.3, Figure 19.3.4). Since about 1996, uo-3 has also been extensively used in cell-based high-throughput screening assays for drug discovery.12,13 Fluo-3 is essentially nonuorescent unless bound to Ca2+ and exhibits a quantum yield at saturating Ca2+ of ~0.14 (Figure 19.3.5) and a Kd for Ca2+ of 390 nM (measured at 22C using our Calcium Calibration Buer Kits). e intact acetoxy-methyl (AM) ester derivative of uo-3 is almost nonuorescent, unlike the AM esters of fura-2 and indo-1. e green-uorescent emission (~525 nm) of Ca2+-bound uo-3 is conventionally detected using optical lter sets designed for uorescein (FITC).

In a careful study of the spectral properties of highly puried uo-3, Harkins, Kurebayashi and Baylor characterized the eects of pH and viscosity on Ca2+ measurements with uo-3 and dem-onstrated that binding of the indicator to proteins has a signicant eect on its Kd for Ca2+ 14 (Table 19.2). e temperature dependence of the Kd for uo-3 has also been reported.15 In addition, the uorescence output of uo-3the product of the molar absorptivity and the uorescence quantum yieldmay also vary signicantly in dierent cellular environments.16,17

Fluo-3 exhibits an at least 100-fold Ca2+-dependent uorescence enhancement.4,14 However, uo-3 lacks a signicant shi in emission or excitation wavelength upon binding to Ca2+, which precludes the use of ratiometric measurements (Figure 19.3.6). Simultaneous loading of cells with uo-3 and our Fura Red indicator, which exhibit reciprocal shis in uorescence intensity upon binding Ca2+, has enabled researchers to make ratiometric measurements of intracellular Ca2+ (Figure 19.3.7) using confocal laser-scanning microscopy 18,19 (Figure 19.3.8) or ow cytom-etry.2022 For ratiometric measurements, uo-3 (or uo-4) can also be co-loaded into cells with a spectrally distinct Ca2+-insensitive dye, such as CellTracker Orange CMRA 23 (C34551, Section 14.2) or CellTrace calcein red-orange AM 24 (C34851, Section 15.2). It is common practice when loading neurons in brain slices via patch pipette infusion with green-uorescent calcium indica-tors, such as uo-3, uo-4, uo-5F and Oregon Green 488 BAPTA-1, to add in a Ca2+-insensitive structural marker such as Alexa Fluor 594 hydrazide (A10438, A10442; Section 14.3).2527

Fluo-3 is available as a cell-impermeant potassium salt (F3715) or ammonium salt (F1240). e cell-permeant uo-3 AM is available as a 1 mg vial (F1241), as a set of 20 vials each containing 50 g (F1242), as a set of 10 vials each containing 50 g of our high-purity grade uo-3 AM (F23915) and as a 1 mM solution in DMSO (F14218). A set of 40 vials, each containing 1 mg of uo-3 AM (F14242), is available at a discounted price to provide a larger quantity for high-throughput screening applications.

Figure 19.3.1 Fluo indicators.

O O

R7'

O

N(CH2CO)2

OOCH2CH2OO

(OCCH2)2N

R7'

R6

R6

R5

R5

Fluo-3

Fluo-4

Fluo-5F

Fluo-5N

Fluo-4FF

0.39 M

0.35 M

2.3 M

90 M

9.7 M

Cl

F

F

F

F

Cl

F

F

F

F

CH3CH3F

NO2F

H

H

H

H

F

Indicator Kd(Ca2+)

R2'

R2'

Figure 19.3.2 Two-photon excitation imaging of Ca2+ inux in a CA1 pyramidal cell spine. The images are overlays of anatomical images generated by Alexa Fluor 594 hydrazide (A10438, A10442) and Ca2+ signals generated by uo-5F (F14221). The imaging system uses two-photon excitation at 810 nm and two-channel emission detection (uo-5F in the green channel, Alexa Fluor 594 hydrazide in the red channel). The observed Ca2+ inux is through NMDA receptors that are activated by glutamate released from the presynaptic terminal following electrode stimulation of a collateral CA3 pyramidal cell axon. The brief (0.2 ms) depolariz-ing stimulus was applied after the rst frame in the image sequence. The frame rate is four frames/second, and each frame repre-sents an area of 5 m 5 m. The image was contributed by Thomas Oertner and Karel Svoboda, Cold Spring Harbor Laboratory.

Figure 19.3.3 Fluo-3 (F1240) confocal image of a spi-ral Ca2+ wave initiated by injection of a nonhydrolyzable analog of inositol 1,4,5-triphosphate in a Xenopus laevis oocyte. The image was contributed by David Clapham, Harvard University, and reproduced with permission from Cell (1992) 69:283.

Figure 19.3.4 Spontaneous intracellular Ca2+ uctuations of neurons developing in vivo. The spinal cord was dissect-ed from a neurula-stage Xenopus embryo and loaded with uo-3 AM (F1241, F1242, F14218, F14242, F23915). Regions of uo-3 uorescence on the ventral side of the spinal cord are pseudocolored in gold and indicate areas of highest in-tracellular Ca2+ (Nature (1995) 375:784). The image was ob-tained with a Bio-Rad MRC600 confocal laser-scanning mi-croscope. The image was contributed by Nicholas C. Spitzer, University o

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