4.15. Flow Cytometry Analysis

HL60, DMSO-treated HL-60, THP-1, and PMA-treated THP-1 cells were seeded at 1.5E5 cells/well on a clear 96-well plate. The cells were washed with 200 µL/well of PBS without Ca²⁺ and Mg²⁺, supplemented with 2% FBS (FACS buffer). For HL60 cells, CD11b-FITC (BDPharmingen; Cat: 562,793 Clone: ICRF44) was added to each well at 1:40 dilution (50 µL) in FACS buffer and incubated at RT, being covered from light, for 15 min. For THP-1 cells, a combination of CD11b and CD14-APC/Cy7 stains (Cat: 301,820 Clone: M5E2) were used to monitor CD11b upregulation in addition to macrophage differentiation. The cells were washed twice with FACS buffer at 1400 rpm for 5 min. each and LIVE/DEAD Fixable Near-IR Dead Cell stain (ThermoFisher Scientific, Waltham, MA, USA) was added to each well at a dilution of 1:500 (50 µL) and incubated on ice, covered from light, for 15 min. The cells were washed again and resuspended in 200 µL FACS buffer. Fluorescence measurements were acquired using either Guava flow cytometer (EMD Millipore, Burlington, MA, USA) or they were acquired at a Symphony A3 (BD Biosciences, Franklin Lakes, NJ USA). Data was analyzed with FlowJo software V10. Induction was considered to be successful if CD11b expression in viable cells was found to be ~70% or higher.