Flow cytometry to analyze CD24 and CD44 expression

MDA-MB-231 cells100 μm were plated and pre-treated with RM or mESC CM for 4 d, after which the media was removed and the cells were washed with PBS. The cells were trypsinized and deactivated with wash buffer (PBS, 1% FBS, 1% Penstrep), spun down for 5 minutes at 1500 rpm, and the supernatant was aspirated. The cells were then re-suspended in 5 mL of wash buffer and counted to determine the volume needed to obtain 1 × 10⁶ cells. The cells were pipetted into an eppendorf tube for each of the two samples, spun down at 1500 rpm for 5 minutes and supernatant was removed. The pellet was re-suspended in 100uL of wash buffer and this solution was kept on ice and kept from light for the rest of the experiment. Next, fluorescently conjugated antibodies for CD44 and CD24 were added directly to the cell solution (20uL CD44-FITC and 20uL CD24-PE to each sample) and the solution was vortexed. In the control samples, 20uL FITC isotype and 20uL PE isotype controls were added. The antibody/cell suspension was incubated in the dark for 30 minutes at 4°C. Following incubation, cells were washed twice in 1mL of wash buffer to remove excess antibody and then the pellet was re-suspended and fixed in 0.5mL of 1% paraformaldehyde and vortexed. Samples were taken directly to the Northwestern Flow Cytometry Core Facility where the samples were analyzed using a Fortessa flow cytometer (BD Biosciences). The spectral overlap between FITC and PE fluorochrome emissions and the subsequent compensation values were calculated via the FACSDiva software (BD Biosciences), from unlabeled and single-antibody (single-color) controls, prior to running test samples.