Apc conjugated cd11b antibody

1×10⁶ cells were collected from cultures and centrifuged at 700 rpm for 5 min. Cell pellets were resuspended in 200 µl 37°C PBS containing 2.5 µl of either APC-conjugated CD11b antibody, PE-conjugated CD38 antibody, or PE-conjugated CD14 antibody (all from BD Biosciences, San Jose, CA). Following 1 h incubation at 37°C, cell surface expression levels were analyzed with a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). APC fluorescence (excitation at 633 nm) was collected with a 660/20 band pass filter and PE fluorescence (excitation at 488 nm) was collected with a 576/26 band pass filter. Undifferentiated control cells were used to determine the fluorescence intensity of cells negative for the respective surface antigen. The gate to determine percent increase of expression was set to exclude 95% of the control population.

For the phenotypic analysis, cultures of HL‐60 cells were initiated at a density of 0.1 × 10⁶ mL on day 0. The cells were treated with either 1 μmol/L RA (Sigma) or 0.5 μmol/L 1,25‐dihydroxyvitamin D3 (Cayman) to induce differentiation. For the staining of CD38, CD11b, and CD14, 1 × 10⁶ cells were collected and centrifuged at 120 × g for 5 minutes. The cell pellets were resuspended in 200 μL PBS with 2.5 μL of APC‐conjugated CD11b antibody, PE‐conjugated CD38 antibody or PE‐conjugated CD14 antibody (all from BD Biosciences) at 37°C for 1 hour and analyzed with an LSR II flow cytometer (Becton Dickinson). For the cell cycle analysis, the same number of cells was centrifuged at 120 g for 5 min, and stained by resuspension in PI solution (50 mg/mL propidium iodine, 1 ml/mL Triton X‐100, and 1 mg/mL sodium citrate), stored at 4°C overnight, and then analyzed by flow cytometry. Gating was set to exclude debris and doublets.