Cell Meter™ Fluorimetric Phagocytosis Assay Kit *Red Fluorescence*

1. Plate cells.

2. Add 12.5 µL Protonex™ Red 600-Latex Bead Conjugates.

3. Incubate at 37 °C for 4 hours.

4. Add 12.5 µL CytoTrace™ Green.

5. Incubate at 37 °C for 30 minutes.

6. Monitor fluorescence by microscopy using Texas Red and FITC filters.

Thaw all the kit components to room temperature before starting the experiment.

For guidelines on cell sample preparation, please visit:

https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Plate cells overnight in a growth medium at 20,000-50,000 cells/well/100 µL in a 96-well plate.

   Note: For the RAW 264.7 cells used in this assay, we recommend plating 50,000 cells per well in 100 µL of medium in a 96-well plate and incubating them overnight. It is important to optimize the cell density for each cell line individually.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

1. Add 8 µL of Protonex™ Red 600-Latex Bead Conjugates (Component A) to 2 mL of cell growth medium (containing 10% FBS), and mix well.

   Note: Unused beads can be stored at 4 °C.

1. Add 20 µL of DMSO (Component C) to the vial of CytoTrace™ Green (Component B) and mix well.

   Note: Unused CytoTrace™ Green DMSO stock solution can be aliquoted into single-use vials and stored at -20 °C, protected from light.

1. Add 5 µL of CytoTrace™ Green stock solution (400X) to 2 mL of cell growth medium and mix well.

1. Add 25 µL of 6X Cytochalasin D (not provided) as a positive control or your test compound into each well. The final concentration in the well should be 10 µM.

   Note: To prepare a 6X Cytochalasin D solution, add 18 µL of 10 mM Cytochalasin D (not included) to 3 mL of cell growth medium, and mix thoroughly. Be sure to optimize the Cytochalasin D concentration for each specific cell line used in the assay.

2. Incubate the plate in the cell incubator for 30 minutes.

3. Add 12.5 µL of the 12X Protonex™ Red 600-Latex Bead Conjugate solution to each well.

4. Incubate the plate in the cell incubator for 4 hours.

   Note: The incubation time should be optimized by users for each individual cell lines.

5. Add 12.5 µL of the 12X CytoTrace™ Green working solution to each well.

6. Incubate the plate in the cell incubator for 30 minutes.

7. Wash the plate twice with 1X PBS.

8. Observe phagocytosis inside the cells with Texas Red filter (Ex/Em = 570/600 nm) and CytoTrace™ Green with FITC filter (Ex/Em = 490/525 nm).