Protonex™ Red 670-E. coli Conjugate

1. Plate the cells.
2. Treat cells with test compounds.
3. Add Protonex Dye E. coli conjugates in medium.
4. Incubate at 37°C for 60 minutes.
5. Monitor fluorescence by microscope or fluorescence plate reader.

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 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.

Note: Higher background fluorescence levels may be seen with poly-D-lysine coated microplates.

Add phagocytosis inhibitor (e.g., Cytochalasin D ) at the desired concentrations. You may need to add vehicle controls to untreated wells. (For example: 11X working solution can be prepared in PBS, and 10 µL can be added to each well.)
Note: The time and concentration of phagocytosis effectors varies with cell types.

1. Add the suspension of E. coli conjugate to the cell culture microplate in a 1:10 dilution, or 10 μL of particles added to 100 μL of cell culture medium, and mix well.
2. Place the cells at 37°C for 60 minutes to 3 hours.

1. Wash the cells 2-3 times with HHBS Buffer (AAT Cat# 20011) or buffer of your choice.
2. Add 100 µL HHBS Buffer to each well.
3. Observe plate with a fluorescence microscope using the following filter set or read plate in a fluorescence plate reader with bottom read mode.