incucyte phagocytosis assay real-time quantitative ...€¦ · quantification of phagocytosis over...
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1 essenbiosciencecomIncuCyte
A simple mix-and-read imaging assay for automated quantification of phagocytosis over time in living cells within your incubator The assay provides real-time visualization and analysis of the internalization of bioparticles using pH sensitive-conjugated probes and is ideal for monitoring phagocytosis of bacterial gram positive gram negative or yeast-derived pathogens by immune cells
Figure 1 Validation of phagocytosis using time-lapse imaging Time-lapse visualization of J774A1 mouse macrophages phagocytosing IncuCytetrade pHrodoreg Green E coli Bioparticlesreg over 4 hours Images verify the presence of fluorescent punctate (phagosomal) labelled structures in the cytosol but not in the nucleus
Figure 2 Quantification of phagocytosis using fluorescence segmentation IncuCytetrade software enables accurate segmentation of the fluorescence image (pink mask) and minimizes the impact of background fluorescence
Measure green fluorescence IncuCytetrade pHrodo Green E coli Bioparticlesreg
Measure red fluorescence IncuCytetrade pHrodo Red S aureus Bioparticlesreg
0 30prime 1 h 2 h 4 h
Key Advantages of the IncuCytetrade Phagocytosis Assaybull Validation of phagocytosis using time-lapse imaging (Figure 1)bull Automated analysis and quantification (Figure 2)bull Mix-and-read 96384-well format no fixing no quenching no
lifting (Figure 3)bull Highly sensitive low background low cell numbers (Figures 4 amp 5)bull Sterile IncuCytetrade pHrodoreg Bioparticlesreg enable long-term
measurements (0 to gt48 hours) in real time
IncuCytetrade Phagocytosis AssayReal-time quantitative analysis of phagocytosis
0001 001 01 1 10 100 10000
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5
Latruculin ACytochalasin D
Nocodazole
[Inhibitor] (microM)
AU
C x
105 (0
-12h
)
0 1 3 1000
04
08
12
Cellswell x103
AU
C x
106 (0
-10h
)
Cell number-dependence(10 microg of Bioparticlesreg)
Bioparticle quantity-dependence of phagocytosis(10K cellswell)
0 3 10 3000
04
08
12
Bioparticle (microgwell)
AU
C x
106 (0
-10h
)
0 2 4 6 8 1000
05
10
15
20
2530 microgwell10 microgwell3 microgwell0 microgwell
Time (hours)
Gre
en O
bjec
t Are
a (micro
msup2 x
105 Im
age)
0 2 4 6 8 1000
05
10
15
20
2510K cellswell3K cellswell1K cellswell0 cellswell
Time (hours)
Gre
en O
bjec
t Are
a (micro
msup2 x
105 Im
age)
2 essenbiosciencecomIncuCyte
Figure 3 IncuCytetrade phagocytosis assay quick start guide
Figure 4 Quantification of phagocytosis is cell number dependent J774A1 mouse macrophages phagocytosing IncuCytetrade pHrodoreg Green S aureus Bioparticlesreg (96-well format) Note high signal background with as low as 3K cells per well
Figure 5 Inhibition of phagocytosis by Cytochalasin D Latruculin A NocodazoleJ774A1 murine macrophages phagocytosing IncuCytetrade pHrodoreg Green E coli Bioparticlesreg in the presence of inhibitors
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SEED TARGET CELLS
TREATCELLS
ADD INCUCYTEtrade PHRODOreg BIOPARTICLESreg FOR PHAGOCYTOSIS
LIVE CELL FLUORESCENT IMAGING
Phagocyte Cell SeedingSeed phagocytes (50 microLwell 1 x103 to 1 x104 cellswell) into the 96-well plate and leave to adhere (2 - 16 h)
ActivatorInhibitor or Molecular InterventionAdd the desired treatments (25 microLwell) at 4x final assay concentrations
IncuCytetrade pHrodoreg Bioparticlesreg AdditionAdd your choice of Bioparticlereg (eg E coli S aureus Zymosan) to the 96-well plate (approximately 10 microg per well depending on Bioparticle 25 microLwell at 4x final assay concentrations)
Automated Imaging and Quantitative AnalysisCapture images every 10-30 minutes (20x or 10x) in IncuCyte ZOOMreg for 2-48 hours Analyze using integrated software
3 essenbiosciencecomIncuCyte
pHrodoreg Bioparticles Added to PhagocytesLittle or no pHrodoreg fluorescence while Bioparticlesreg remain in the pH 74 extracellular environment
pH 74
pH 74pH 45 -55
Phagocytosis Initiated Following Receptor ActivationFormation of the phagocytic cup
Formation of the PhagosomeEngulfment of Bioparticlesreg by pinching off The acidic environment of the phagosome (pH 45-55) leads to increased pHrodoreg fluorescence
Assay Concept 1 Macrophages (eg J774A1 mouse
macrophages) or other phagocytic cells are seeded in 96- or 384- well micro-titer plates
2 Phagocytosis is measured in real-time by adding mix-and-read sterile IncuCytetrade pHrodoreg Bioparticlesreg
3 An increase in cellular fluorescence indicates the internalization of Bioparticlesreg into the phagosome IncuCytetrade automated image analysis enables quantitation of phagocytosis over time
IncuCytetrade Phagocytosis Assay vs Other Common Approaches Common methods used to assess phagocytosis are often end-point (eg High Content Analysis (HCA)) require cell lifting (eg flow cytometry) or washingquenching of the labelled pathogen (eg fluorescein labelled)
Reader IncuCytetrade ZOOM Plate reader
Flow cytometry HCA ELISA
Real-time cell visualization
Integrated analysis
Mix-and-read no wash or quench
Access long term phagocytosis gt12h
Flexible choice of effector cells
No fixationlifting
High sensitivitylow background
Low cell numberbioparticle number
321
bull Real-time imaging and quantitation of chemotactic cell migration and invasion for label-free or fluorescently labeled cells from within your incubator
bull See everything with high definition images including morphological changes cell-cell interactions and collective cell migration and invasion
bull Highly reproducible kinetic read-outs compatible with detailed mechanistic studies andor 96-well screening and profiling
Learn more at essenbiosciencecomchemotaxis
RELATED APPLICATIONS
Chemotactic Cell Migration and Invasion Using the IncuCyte ZOOMreg System
4 essenbiosciencecomIncuCyte8000-0368-D00
Ordering information
Product Quantity Cat No
IncuCytetrade pHrodoreg Red E coli Bioparticlesreg for Phagocytosis 2 mg 4615
IncuCytetrade pHrodoreg Green E coli Bioparticlesreg for Phagocytosis 2 mg 4616
IncuCytetrade pHrodoreg Red Zymosan Bioparticlesreg for Phagocytosis 1 mg 4617
IncuCytetrade pHrodoreg Green Zymosan Bioparticlesreg for Phagocytosis 1 mg 4618
IncuCytetrade pHrodoreg Red S aureus Bioparticlesreg for Phagocytosis 2 mg 4619
IncuCytetrade pHrodoreg Green S aureus Bioparticlesreg for Phagocytosis 2 mg 4620
Learn more at essenbiosciencecomapplications
Other Key Applications Using the IncuCyte ZOOMreg Live-Cell Imaging System
Essen BioScience 300 West Morgan Road Ann Arbor Michigan USA 48108 copy 2016 Essen BioScience All rights reserved All trademarks are the property of Essen BioScience unless otherwise specified
3D-Spheroids Angiogenesis Apoptosis Cell Culture QC
Dilution Cloning
Cytotoxicity
Neurite Dynamics - Label-Free
Neuronal Co-Culture - Fluorescence
Proliferation - Cell Count
Proliferation - Confluence
Reporter Gene Transfection Efficiency
Stem Cell Monitoring amp Reprogramming
Immune Cell Killing Clustering amp Proliferation
Scratch Wound Migration amp Invasion
ChemotaxisMigration amp Invasion
Learn more at essenbiosciencecomphagocytosis
Phagocytosis
0001 001 01 1 10 100 10000
1
2
3
4
5
Latruculin ACytochalasin D
Nocodazole
[Inhibitor] (microM)
AU
C x
105 (0
-12h
)
0 1 3 1000
04
08
12
Cellswell x103
AU
C x
106 (0
-10h
)
Cell number-dependence(10 microg of Bioparticlesreg)
Bioparticle quantity-dependence of phagocytosis(10K cellswell)
0 3 10 3000
04
08
12
Bioparticle (microgwell)
AU
C x
106 (0
-10h
)
0 2 4 6 8 1000
05
10
15
20
2530 microgwell10 microgwell3 microgwell0 microgwell
Time (hours)
Gre
en O
bjec
t Are
a (micro
msup2 x
105 Im
age)
0 2 4 6 8 1000
05
10
15
20
2510K cellswell3K cellswell1K cellswell0 cellswell
Time (hours)
Gre
en O
bjec
t Are
a (micro
msup2 x
105 Im
age)
2 essenbiosciencecomIncuCyte
Figure 3 IncuCytetrade phagocytosis assay quick start guide
Figure 4 Quantification of phagocytosis is cell number dependent J774A1 mouse macrophages phagocytosing IncuCytetrade pHrodoreg Green S aureus Bioparticlesreg (96-well format) Note high signal background with as low as 3K cells per well
Figure 5 Inhibition of phagocytosis by Cytochalasin D Latruculin A NocodazoleJ774A1 murine macrophages phagocytosing IncuCytetrade pHrodoreg Green E coli Bioparticlesreg in the presence of inhibitors
1
2
3
4
SEED TARGET CELLS
TREATCELLS
ADD INCUCYTEtrade PHRODOreg BIOPARTICLESreg FOR PHAGOCYTOSIS
LIVE CELL FLUORESCENT IMAGING
Phagocyte Cell SeedingSeed phagocytes (50 microLwell 1 x103 to 1 x104 cellswell) into the 96-well plate and leave to adhere (2 - 16 h)
ActivatorInhibitor or Molecular InterventionAdd the desired treatments (25 microLwell) at 4x final assay concentrations
IncuCytetrade pHrodoreg Bioparticlesreg AdditionAdd your choice of Bioparticlereg (eg E coli S aureus Zymosan) to the 96-well plate (approximately 10 microg per well depending on Bioparticle 25 microLwell at 4x final assay concentrations)
Automated Imaging and Quantitative AnalysisCapture images every 10-30 minutes (20x or 10x) in IncuCyte ZOOMreg for 2-48 hours Analyze using integrated software
3 essenbiosciencecomIncuCyte
pHrodoreg Bioparticles Added to PhagocytesLittle or no pHrodoreg fluorescence while Bioparticlesreg remain in the pH 74 extracellular environment
pH 74
pH 74pH 45 -55
Phagocytosis Initiated Following Receptor ActivationFormation of the phagocytic cup
Formation of the PhagosomeEngulfment of Bioparticlesreg by pinching off The acidic environment of the phagosome (pH 45-55) leads to increased pHrodoreg fluorescence
Assay Concept 1 Macrophages (eg J774A1 mouse
macrophages) or other phagocytic cells are seeded in 96- or 384- well micro-titer plates
2 Phagocytosis is measured in real-time by adding mix-and-read sterile IncuCytetrade pHrodoreg Bioparticlesreg
3 An increase in cellular fluorescence indicates the internalization of Bioparticlesreg into the phagosome IncuCytetrade automated image analysis enables quantitation of phagocytosis over time
IncuCytetrade Phagocytosis Assay vs Other Common Approaches Common methods used to assess phagocytosis are often end-point (eg High Content Analysis (HCA)) require cell lifting (eg flow cytometry) or washingquenching of the labelled pathogen (eg fluorescein labelled)
Reader IncuCytetrade ZOOM Plate reader
Flow cytometry HCA ELISA
Real-time cell visualization
Integrated analysis
Mix-and-read no wash or quench
Access long term phagocytosis gt12h
Flexible choice of effector cells
No fixationlifting
High sensitivitylow background
Low cell numberbioparticle number
321
bull Real-time imaging and quantitation of chemotactic cell migration and invasion for label-free or fluorescently labeled cells from within your incubator
bull See everything with high definition images including morphological changes cell-cell interactions and collective cell migration and invasion
bull Highly reproducible kinetic read-outs compatible with detailed mechanistic studies andor 96-well screening and profiling
Learn more at essenbiosciencecomchemotaxis
RELATED APPLICATIONS
Chemotactic Cell Migration and Invasion Using the IncuCyte ZOOMreg System
4 essenbiosciencecomIncuCyte8000-0368-D00
Ordering information
Product Quantity Cat No
IncuCytetrade pHrodoreg Red E coli Bioparticlesreg for Phagocytosis 2 mg 4615
IncuCytetrade pHrodoreg Green E coli Bioparticlesreg for Phagocytosis 2 mg 4616
IncuCytetrade pHrodoreg Red Zymosan Bioparticlesreg for Phagocytosis 1 mg 4617
IncuCytetrade pHrodoreg Green Zymosan Bioparticlesreg for Phagocytosis 1 mg 4618
IncuCytetrade pHrodoreg Red S aureus Bioparticlesreg for Phagocytosis 2 mg 4619
IncuCytetrade pHrodoreg Green S aureus Bioparticlesreg for Phagocytosis 2 mg 4620
Learn more at essenbiosciencecomapplications
Other Key Applications Using the IncuCyte ZOOMreg Live-Cell Imaging System
Essen BioScience 300 West Morgan Road Ann Arbor Michigan USA 48108 copy 2016 Essen BioScience All rights reserved All trademarks are the property of Essen BioScience unless otherwise specified
3D-Spheroids Angiogenesis Apoptosis Cell Culture QC
Dilution Cloning
Cytotoxicity
Neurite Dynamics - Label-Free
Neuronal Co-Culture - Fluorescence
Proliferation - Cell Count
Proliferation - Confluence
Reporter Gene Transfection Efficiency
Stem Cell Monitoring amp Reprogramming
Immune Cell Killing Clustering amp Proliferation
Scratch Wound Migration amp Invasion
ChemotaxisMigration amp Invasion
Learn more at essenbiosciencecomphagocytosis
Phagocytosis
3 essenbiosciencecomIncuCyte
pHrodoreg Bioparticles Added to PhagocytesLittle or no pHrodoreg fluorescence while Bioparticlesreg remain in the pH 74 extracellular environment
pH 74
pH 74pH 45 -55
Phagocytosis Initiated Following Receptor ActivationFormation of the phagocytic cup
Formation of the PhagosomeEngulfment of Bioparticlesreg by pinching off The acidic environment of the phagosome (pH 45-55) leads to increased pHrodoreg fluorescence
Assay Concept 1 Macrophages (eg J774A1 mouse
macrophages) or other phagocytic cells are seeded in 96- or 384- well micro-titer plates
2 Phagocytosis is measured in real-time by adding mix-and-read sterile IncuCytetrade pHrodoreg Bioparticlesreg
3 An increase in cellular fluorescence indicates the internalization of Bioparticlesreg into the phagosome IncuCytetrade automated image analysis enables quantitation of phagocytosis over time
IncuCytetrade Phagocytosis Assay vs Other Common Approaches Common methods used to assess phagocytosis are often end-point (eg High Content Analysis (HCA)) require cell lifting (eg flow cytometry) or washingquenching of the labelled pathogen (eg fluorescein labelled)
Reader IncuCytetrade ZOOM Plate reader
Flow cytometry HCA ELISA
Real-time cell visualization
Integrated analysis
Mix-and-read no wash or quench
Access long term phagocytosis gt12h
Flexible choice of effector cells
No fixationlifting
High sensitivitylow background
Low cell numberbioparticle number
321
bull Real-time imaging and quantitation of chemotactic cell migration and invasion for label-free or fluorescently labeled cells from within your incubator
bull See everything with high definition images including morphological changes cell-cell interactions and collective cell migration and invasion
bull Highly reproducible kinetic read-outs compatible with detailed mechanistic studies andor 96-well screening and profiling
Learn more at essenbiosciencecomchemotaxis
RELATED APPLICATIONS
Chemotactic Cell Migration and Invasion Using the IncuCyte ZOOMreg System
4 essenbiosciencecomIncuCyte8000-0368-D00
Ordering information
Product Quantity Cat No
IncuCytetrade pHrodoreg Red E coli Bioparticlesreg for Phagocytosis 2 mg 4615
IncuCytetrade pHrodoreg Green E coli Bioparticlesreg for Phagocytosis 2 mg 4616
IncuCytetrade pHrodoreg Red Zymosan Bioparticlesreg for Phagocytosis 1 mg 4617
IncuCytetrade pHrodoreg Green Zymosan Bioparticlesreg for Phagocytosis 1 mg 4618
IncuCytetrade pHrodoreg Red S aureus Bioparticlesreg for Phagocytosis 2 mg 4619
IncuCytetrade pHrodoreg Green S aureus Bioparticlesreg for Phagocytosis 2 mg 4620
Learn more at essenbiosciencecomapplications
Other Key Applications Using the IncuCyte ZOOMreg Live-Cell Imaging System
Essen BioScience 300 West Morgan Road Ann Arbor Michigan USA 48108 copy 2016 Essen BioScience All rights reserved All trademarks are the property of Essen BioScience unless otherwise specified
3D-Spheroids Angiogenesis Apoptosis Cell Culture QC
Dilution Cloning
Cytotoxicity
Neurite Dynamics - Label-Free
Neuronal Co-Culture - Fluorescence
Proliferation - Cell Count
Proliferation - Confluence
Reporter Gene Transfection Efficiency
Stem Cell Monitoring amp Reprogramming
Immune Cell Killing Clustering amp Proliferation
Scratch Wound Migration amp Invasion
ChemotaxisMigration amp Invasion
Learn more at essenbiosciencecomphagocytosis
Phagocytosis
4 essenbiosciencecomIncuCyte8000-0368-D00
Ordering information
Product Quantity Cat No
IncuCytetrade pHrodoreg Red E coli Bioparticlesreg for Phagocytosis 2 mg 4615
IncuCytetrade pHrodoreg Green E coli Bioparticlesreg for Phagocytosis 2 mg 4616
IncuCytetrade pHrodoreg Red Zymosan Bioparticlesreg for Phagocytosis 1 mg 4617
IncuCytetrade pHrodoreg Green Zymosan Bioparticlesreg for Phagocytosis 1 mg 4618
IncuCytetrade pHrodoreg Red S aureus Bioparticlesreg for Phagocytosis 2 mg 4619
IncuCytetrade pHrodoreg Green S aureus Bioparticlesreg for Phagocytosis 2 mg 4620
Learn more at essenbiosciencecomapplications
Other Key Applications Using the IncuCyte ZOOMreg Live-Cell Imaging System
Essen BioScience 300 West Morgan Road Ann Arbor Michigan USA 48108 copy 2016 Essen BioScience All rights reserved All trademarks are the property of Essen BioScience unless otherwise specified
3D-Spheroids Angiogenesis Apoptosis Cell Culture QC
Dilution Cloning
Cytotoxicity
Neurite Dynamics - Label-Free
Neuronal Co-Culture - Fluorescence
Proliferation - Cell Count
Proliferation - Confluence
Reporter Gene Transfection Efficiency
Stem Cell Monitoring amp Reprogramming
Immune Cell Killing Clustering amp Proliferation
Scratch Wound Migration amp Invasion
ChemotaxisMigration amp Invasion
Learn more at essenbiosciencecomphagocytosis
Phagocytosis