Cell Meter™ Fluorimetric Phagocytosis Assay Kit *Green Fluorescence*

1. Plate cells.

2. Add Protonex™ Green 500-Zymosan Beads Conjugate solution.

3. Incubate at 37 °C for 60 minutes.

4. Add CytoTrace™ Red.

5. Incubate at 37 °C for 30 minutes.

6. Monitor fluorescence by microscopy using FITC and Texas Red 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 20 µL of DMSO (Component C) to the vial containing CytoTrace™ Red (Component B), and mix thoroughly.
   
   Note: Please prepare the stock solution before use. Unused 400X CytoTrace™ Red stock solution in DMSO can be aliquoted into single-use vials and stored at -20 °C, protected from light

1. Add 150 µL of Protonex™ Red 500-Zymosan Beads Conjugates (Component A) to 1.5mL of cell growth medium (containing 10% FBS), and mix well.

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

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

   Note: Please prepare the stock solution before use.

1. For negative control, add 25 µL of 6X Cytochalasin D (not provided). The final concentration in the well should be 10 µM. Similarly, for positive control, add LPS to a final concentration of 250ng/mL. Prepare your test compounds and add to the wells.

   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 and LPS concentration for each specific cell line used in the assay.

   Note: Cytochalasin D and LPS are not provided in the kit.

2. Incubate the plate in the cell incubator for 30 minutes for Cytochalasin and for 8 hours or more for LPS treatment. 

3. Add 12.5 µL of the 12X Protonex™ Green 500-Latex Bead Conjugate solution to each well.

4. Incubate the plate in a cell incubator for 60 minutes.

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

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

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

7. Wash the plate twice with 1X PBS.

8. Monitor phagocytosis within the cells using the FITC filter (Ex/Em= 490/525 nm) for green fluorescence, and observe CytoTrace™ Red using the Texas Red filter (Ex/Em = 570/600 nm) for red fluorescence.