Macrophage phagocytosis assay using fluorescence microscope and flow cytometer

The phagocytosis ability of macrophages was quanified using EGFP-expressing E. coli as the previous literature described [10]. To be brief, add 100 μL of fresh culture medium and 10 μL of EGFP-expressing E. coli. Suspension (approximately 2 × 10⁷ cells) into each well of a 24-well plate which pre-cultured the peritoneal macrophages. Place the plate in a CO2 incubator for 1 h to allow the macrophages to phagocytize the bacteria. Firstly, wash once with 0.8% crystal violet water solution to quench the fluorescence of non-internalized bacteria. After washing with cold PBS, fix the cells with 4% formaldehyde at RT for 30 min. Then, wash with PBS and incubate the cells at RT for 60 min in dark with phalloidin-Alexa Fluor 633 conjugate working solution to stain the F-actin. Wash the cells with PBS and then incubate with DAPI 5 min at RT to stain the cell nuclear. Rinse once with PBS and observe using an invert fluorescence microscope (Leica DMI-3000). Count the green-fluorescence-positive cells in 10 randomly selected areas, and calculate the percentages of these cells using FIJI (ImageJ) software version 2.0 (NIH, USA).

The flow cytometer (BD FACS Canto-II) was used to quantify the phagocytosis ability of the macrophages precisely. The primary macrophages were cultured in a 6-well plate. Add 50 μL of EGFP-expressing E. coli. Suspension (approximately 1 × 10⁸ cells) into the wells according to the group setting. Then place the 6-well plate in the 37 °C 5% CO2 incubator for 1 h. Wash with 0.8% CV working solution shortly to quench the fluorescence of extracellular bacteria. Wash the cells with PBS 3 times to remove any residual CV. Add 70 mM cold EDTA to detach the cells and transfer into the flow cytometry tube. Use F4/80-PE conjugated antibody (Biolegend) to mark the macrophages. Resuspend the cell pellets with 200–300 μL of PBS for flow cytometry analysis. Run each tube and acquire data for at least 10,000 events of F4/80-positive cells. BD FACS-Diva software (Version 8.0.1) was used to analyze the data and generate the contour plots.