Page 189 - Live-cellanalysis handbook

IncuCyte pHrodo Orange Phagocytosis Assay
®
®

For quantification of phagocytosis of apoptotic and non-apoptotic cell activity

This protocol is intended for the measurement of both apoptotic method utilizes the IncuCyte® pHrodo® Orange Cell Labeling Kit
(efferocytosis) and non-apoptotic phagocytosis (antibody- and the IncuCyte® S3 Live-Cell Analysis System for Neuroscience
dependent cellular phagocytosis) of cells by macrophages. This for image-based fluorescent measurements of phagocytosis.

Required materials
• IncuCyte® pHrodo® Orange Cell • Effector cells of interest
Labeling Kit for Phagocytosis • Effector cell culture media
(Sartorius Cat # 4766) • 96-well microplate (e.g., Corning®
• Target cells of interest Cat # 3595)
• Target cell culture media

Initial Optimization Experiment

1. Assay Optimization 2.3 Perform a serial dilution of the IncuCyte pHrodo Orange Cell
For optimal assay results, conduct preliminary experiment to Labeling Dye in DMSO.
determine the following assay parameters: a. For cells extracted from blood or tissue, generate a
1.1 The seeding density of the effector cells which will result in concentration range between 1 mg/mL (stock) and
10-20% confluence 24 hours after plating. We have found 100 μg/mL.
that 10,000 effector cells per well is a reasonable starting b. For cultured cell lines, generate a concentration range
point to reach ~20% cell confluency, and recommend between 100 μg/mL and 10 μg/mL.
optimizing above and below that density (e.g., 6, 7, 8, 9, 10, 2.4 Add 10 μl of each concentration of dye to 1 mL cell
11, 12 and 13k cells/well). suspension i.e., a 1:100 dilution, which will provide a final
1.2 The lowest concentration of drug treatment (e.g., assay concentration range of
camptothecin or staurosporine) that will induce target cell a. 0 μg/mL to 1 μg/mL,
apoptosis with limited cellular debris following a 24-hour b. 1 μg/mL to 100 ng/mL
exposure. Target cell apoptosis can be measured using the 2.5 Incubate for 1 hour at 37 °C. Harvest cells by centrifugation
IncuCyte® Annexin V Reagent (Cat. No. 4759). for 7 minutes at 1000 rpm.
2.6 Aspirate supernatant and wash cell pellet with 1 mL complete
2. Target Cell Labeling Optimization media (cell type appropriate). Harvest cells by centrifugation
Target cells in the IncuCyte® pHrodo Orange Phagocytosis for 7 minutes at 1000 rpm, aspirate supernatant and
Assay must be efficiently labeled in order to detect phagocytic resuspend in 1 mL complete media.
events. We recommend performing a serial dilution of the 2.7 Prepare a citrate-based buffer solution at pH 4.0. For each
IncuCyte®pHrodo® Orange Cell Labeling Dye in DMSO and labeling dilution of IncuCyte pHrodo Orange Labeled Cells, prepare a
your target cells per the optimization protocol below: micro-centrifuge tube containing 300 μL of buffer, and add
2.1 Suspend target cells at a density of 1 x106 cells/mL in 30 μL of labeled cells. Mix by trituration.
IncuCyte® pHrodo® Labeling Buffer (component D). Separate 2.8 Per each buffered cell dilution, aliquot 100 μL to three
the suspension into aliquots of 1mL. wells of a 96-well plate and allow the cells to settle at
2.2 Solubilize the IncuCyte pHrodo Orange Cell Labeling Dye ambient temperature. Scan the plate in phase and orange
(component A) by adding 100 μl of DMSO (component B) to fluorescence. By counting the number of phase and
create a stock concentration of 1 mg/mL. fluorescent objects, a percentage of labeled cells may be
obtained for each concentration of dye.

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