calcium assays for the flipr system | molecular devices

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Calcium assays for the FLIPR system

DATA SHEET

IntroductionThe FLIPR® Calcium Assay Kits have been the premier choice for measuring changes in intracellular calcium during drug discovery and research. Built as the most comprehensive calcium portfolio, the FLIPR and QBT™ Calcium Assay Kits deliver preoptimized, homogeneous, fluorescence-based formulations to expedite assay development and screening of GPCR and ion channel targets. We have many different kits and sizing options to meet all your research needs.

Optimized homogeneous assaysThis generous portfolio offers five formulations to surmount even your most difficult assays, including frozen, primary or stem cells; transient, endogenous or low-expression receptors; plus chemokines, allosteric modulators or partial agonists. Formulations match a unique fluorophore with our proprietary masking technology, providing optimal conditions for your target of interest (see ‘No-wash workflow’ on Page 2).

KEY FEATURES• Optimized no-wash assays

• Compatible with endogenous, primary or stem cell targets

• Rapid assay development

• Patented quench technology

• Largest signal window available

• Quench- or probenecid-free protocols

FLIPR system

Rapid assay developmentUtilizing a mix-and-read procedure, cells are incubated with the assay kit and then transferred directly to the instrument for evaluation. No intermediate wash steps are required; thus, potential cell detachment, lowered cell competency and incomplete dye removal are eliminated. Protocols are pre-validated for use on the FLIPR® High-Throughput Cellular Screening System to conserve your time while quickly advancing future discoveries. This process will reduce your work by about 40%.

The FLIPR Calcium 6 and 6-QF Assay Kits, utilize a novel fluorescent indicator dye technology to provide the largest signal window on the market. They enable researchers to:

• Rekindle low signal screens; including endogenous, primary or stem cell targets

• Improve assay quality, with or without masking dye technology

• Multiplex assays using a quench-free option to better characterize signal pathways

• Study target behavior in the presence of organic anion transporters using a probenecid optional protocol

Proprietary chemistry and formulation

Increase in cytosolic Ca2+ can be detected by the FLIPR system using calcium-sensitive dye indicators. Each calcium assay kit utilizes a unique calcium sensitive dye that is absorbed into the cell’s cytoplasm during incubation. Upon target activation, intracellular calcium is released and binds to the dye, which increases fluorescence intensity. Molecular Devices calcium kits utilize an extracellular masking technology to block background fluorescence and increase your assay’s signal window. FLIPR Calcium 6-QF Assay Kit is the key exception, as it enables high assay performance without a masking technology for quench-sensitive applications.

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

sensitivedye

buffer

ligand binds to cell-surface receptor

ligand

Significantly reduce fluorescencebackground with one-step protocol

For quench-sensitive targets and multiplexing

Standard Quench-Free

Increase in cytosolic Ca2+ can be detected by FLIPR or FlexStation Systems using calcium-sensitive dye indicators

receptor

Ca2+

sensitive dye

maskingdye

Active Gq

No-wash workflow for high-throughput assays

Component B bu�er Component A vial

Dye-loading solution

Microplate

Add dye-loading solution to microplate and incubate

Run calciumflux assay onFLIPR system

Easy protocol setup. The FLIPR Calcium Assay Kits reduce assay time for 20 plates from ~ five hours to three hours.

FLIPR Calcium 6 Assay Kit provides best-in-class data

HeLa Cells

-4 -3 -2 -1 0 1 2

0

1

2

3

4

5

6Calcium 6

Calcium 5

Fluo-4 Direct

Calcium 6-QF

Log [histamine] µM

ΔF

/F (m

ax-m

in)

-5 -4 -3 -2 -1 0 1

0

1

2

3

4

5Calcium 6

Calcium 6-QF

Calcium 5

Fluo-4 Direct

4 µM Fluo-4 Wash

Ca 6 Ca6-QF Ca 5 Fluo-4 Direct 4 uM Fluo-4W

EC50 (nM) 16 17 23 20 21

Z @ EC 80 0.88 0.84 0.89 0.84 0.71

Signal window 4 4.2 2.7 2.3 1.4

Log [carbachol] µM

ΔF

/F (m

ax-m

in)

FLIPR Calcium 6 Assay Kit: highest signal-to-noise ratio. Comparison of FLIPR Calcium 6 Assay Kits to other calcium indicators was measured using agonism of the muscarinic receptor on CHO M1 cells (left) and agonism of endogneous histamine H1 receptor on HeLa cells (right).

FLIPR Calcium 6 Assay Kit is the best solution for probenecid-sensitive assays

Anion exchange protein resistance. Our novel fluorophore is more resistant to organic anion exchange proteins, and enables FLIPR Calcium 6 Assay Kit to produce a stronger signal in the absence of probenecid, while conserving EC50 values and Z-factors at EC80 > 0.5. Data for CHO-M1 cells are pictured.

-5 -4 -3 -2 -1 0 1

0

1

2

3

4

5

Calcium 6 Kit PBX

Calcium 5 Kit PBX

Calcium 6 Kit no PBX

Calcium 5 Kit no PBX

Ca6+PBX Ca6 (no PBX) Ca5+PBX Ca5 (no PBX)

EC 50 (nM) 13 39 14 ND

Z @ EC 80 0.9 0.86 0.92 ND

CHO-M1 cells in media: probenecid comparison

Log [carbachol] µM

ΔF

/F (m

ax-m

in)

FLIPR Calcium 5 Assay Kit uses a different formulation enabling shorter incubation time

FLIPR Calcium 5 Assay Kit signal window comparison in hTRPV1-HEK-293. The ChanTest hTRPV1-HEK-293 cell line is stably transfected with a constitutively expressed human TRPV1 transient receptor potential channel. Cells were incubated with dye for one hour, then ligand was added during detection on the FLIPR system to cause an influx of calcium and an increase in fluorescence.

Capsaicin CRC

-12 -11 -10 -9 -8 -7 -6 -5

0

10000

20000

30000

Calcium 4 Kit

Calcium 5 Kit

Fluo-4 Direct

[Capsaicin] M

RF

U (m

ax-m

in)

Capsaicin Calcium 4 Calcium 5 Fluo-4 Direct

EC50

(nM) 2.41 2.10 3.19

Z @ EC50

0.64 0.72 0.63

Fura-2 ratiometric option for automated data normalization

5000

4000

3000

2000

1000

-1000

-2000

-3000

0

0 5 10 15 20 25 30 35

Time (sec.)

Re

lativ

e fl

uo

resc

en

ce u

nits

(RF

U)

40 45 50 55 60 65 70 75 80

Decrease in unbound dye (380 nm)

Increase in Ca2+-bound dye (340 nm)

30

Time (sec.)

34

0/3

80

rat

io

35 40 45 50 55 60 65 70 7525201510500

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Fura-2 QBT Calcium Kit ratiometric analysis. Fura-2 QBT Dye is excited at 340 nm and 380 nm, with emission at 510 nm. Upon calcium flux, the cytoplasm’s calcium concentration increases, causing the fluorescence intensity of calcium-bound Fura-2 at 340/510 nm to increase proportionately. In contrast, the 380/510 nm intensity declines in parallel with the diminishing concentration of unbound dye. By ratioing signal traces from the two wavelengths, artifacts associated with uneven dye distribution and cell number variation are minimized because they affect both measurements equally.

Agonism of the muscarinic M1 receptor in CHO-M1 cells

Carbachol Fura-2 QBT BD Kit Fura-2 Wash

EC50

(µM) 0.013 0.017 0.019

Z @ EC80

0.7 0.5 0.38

Window 1.1 0.85 0.7

Agonism of the Muscarinic M1receptor on CHO M1 cells

34

0/3

80

nm

rat

io (m

ax.-m

in.)

Log [carbachol] µM-5 -4 -3 -2 -1 0 1

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4Fura-2 QBT

BD Kit

Fura-2 Wash

The Fura-2 QBT Calcium Kit provides the largest 340/380 nm ratio signal window. This is a direct comparison to BD Ratiometric Calcium Kit and the traditional Fura-2 wash assay.

Antagonism of the histamine H1 receptor in HeLa cells

Fura-2 QBT Calcium Kit comparison: HeLa Cells

-4 -3 -2 -1 0 1 20.0

0.2

0.4

0.6

0.8

1.0

1.2

Fura-2 QBT

BD Kit

Fura-2 Wash

Pyrilamine Fura-2 QBT BD Kit Fura-2 Wash

IC50

(µM) 4.7 2.7 1.3

Z @ IC80

0.8 0.49 0.33

Window 1.1 0.52 0.37

Log [pyrilamine] µM

34

0/3

80

nm

rat

io (m

ax.-m

in.)

Fura-2 QBT Calcium Kit comparison: HEK-293 cells Muscarinic M3 carbachol agonism

-3 -2 -1 0 1 20.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Fura-2 QBT

BD Kit

Fura-2 Wash

Log [carbachol] µM

34

0/3

80

nm

rat

io (m

ax.-m

in.)

Carbachol Fura-2 QBT BD Kit Fura-2 Wash

EC50

(µM) 5.3 4.4 6.8

Z @ EC80

0.77 0.74 0.65

Window 1.4 0.9 0.76

Product Description Part number

FLIPR Calcium 6& 6-QF AssayEvaluation Kit

Calcium 6: 2 vials/kit + Component B Buffer(sufficient for 2 plates; 1 vial = 1 plate)

Calcium 6-QF: 2 vials/kit +Component B Buffer(sufficient for 2 plates; 1 vial = 1 plate)

R8194

FLIPR Calcium 4, 5, 6 Assay Evaluation Kit

3 vials/kit + Component B Buffer(sufficient for 3 plates; 1 vial = 1 plate)

R8172

Fura-2 QBT Calcium Evaluation Kit

2 vials/kit(sufficient for 2 plates; 1 vial = 1 plate)

R6139*

Solutions for all your calcium assay needs

Your need Our solution

I have probenecid-sensitive assay FLIPR Calcium 6 Assay Kit

I need short incubation times FLIPR Calcium 5 Assay Kit

I need ratiometric data Fura-2 QBT Calcium Kit

Deciding on the right kit for your assay?


FLIPR Calcium 6 Assay Explorer Kit


R8190

FLIPR Calcium 6-QF Assay Explorer Kit


R8192



R8185



R8142

Fura-2 QBT CalciumExplorer Kit


R8197*

Optimizing your assay for your target?


FLIPR Calcium 6 Assay Bulk Kit

10 vials/kit(sufficient for 100 plates; 1 vial = 10 plates)

R8191

FLIPR Calcium 6-QF AssayBulk Kit


R8193



R8186



R8141

Fura-2 QBT Calcium Bulk Kit


R8198*

Need medium to large screens?


FLIPR Calcium 6 Assay Express Kit


R8195

FLIPR Calcium 6-QF Assay Express Kit


R8196



R8187



R8143

Ready for fully automated screens?


FLIPR Pipette Tips Black, non-sterile, 96-well50 racks/case

9000-0762

FLIPR Pipette Tips Clear, non-sterile, 96-well50 racks/case

9000-0761

FLIPR Pipette Tips Black, non-sterile, 384-well50 racks/case

9000-0764

FLIPR Pipette Tips Clear, non-sterile, 384-well50 racks/case

9000-0763

Running low on FLIPR tips?

* If you are interested in the Fura-2 QBT Calcium Kit, it will require the following optics: FLIPR Fura-2 Optics Kit part number: 0200-6271

Includes the 335-345 nm LED module, 380–390 nm LED module, and the 475–535 emission filter for the FLIPR system

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calcium assays for the flipr system | molecular devices

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