Flow Cytometry of IPS Celllines

MACS purification and flow cytometry analysis were performed as described in Sharma, et al., 2022). Purified differentiated iPSC cell pellets were washed with FACS wash buffer (2% FBS in DPBS without Ca2+/Mg2+) and fixed in 1 mL of 4% PFA (in DPBS) for 15 min at RT. After incubation, 5 mL of FACS wash buffer were added, and cells were spun down at 400g for 4 min. Cells were resuspended in wash buffer and added to 96 well plate (about 500K/well). The plate was spun down at 400g for 5 min and the supernatant was carefully removed from the plate.Cells were incubated with anti-PMEL17 (RPE progenitor/committed marker, 1:25, Dako/Agilent, #M0634) or anti-TRA-1-81 (iPSC marker, 1:12.5, BD BioScience, #56012) in 50 ul of permeabilization buffer (2% FBS & 0.02% Triton-X-100 in DPBS) overnight at 4 degrees C on a rocking shaker. Isotype control for primary antibody (purified mouse IgG1, 1:25, BioLegend #401402) was used to set a baseline for analysis. Following overnight incubation, each well was washed with 100 ul of wash buffer at 400g for 5 min. The supernatant was carefully removed and cells were incubated with secondary antibody (Alexa Fluor 488, 1:500, Invitrogen #A-21202 or Alexa Fluor 647, 1:500, Invitrogen #A-31571) in 50 ul of permeabilization buffer for 30 min at RT. Then, each well was washed with 100 ul of wash buffer at 400g for 5 min. After removing the supernatant, 150 ul of wash buffer was added to each well for the flow cytometry analysis. The flow cytometry was performed on CytoFLEX S cytometer (Beckman Coulter Inc., Flow Cytometry Core at Children's Hospital of Philadelphia) and data was analyzed using CytExpert software (Beckman Coulter, Inc.).