Measuring Macrophage Phagocytosis in Diabetic Milieu

Raw 264.7 cells were obtained from the ATCC (Manassas, VA). The cells were preincubated for 48 hr at 37°C and 5% CO₂ under: a. Normal glucose (NG), in DMEM; b. Diabetic milieu (DM) as previously described (29 (link)); c. DM + AII (100 nM); d. DM + AII (Bachem) (100 nM) and A(1-7) (100 nM); e. DM + AII (100 nM) and AVE 0991 (MedChem Express, Princeton, NJ) (10 nM); f. DM + AII (100 nM) and AVE 0991 (100 nM); g. DM + AII (100 nM) and AVE 0991 (1000 nM). Next, the cells were harvested, incubated for 1 h with pHrodo and fixed with 4% formaldehyde solution. Mean fluorescence intensity was determined by flow cytometry using a BD™ LSR II flow cytometer.