Falcon 96 well 5 bottom plates

80% confluent cells were detached from the plates into single cell suspension using Accutase (StemCell Technologies, Cat. # 07920). Cells were washed in DPBS, then fixed with 4% paraformaldehyde in PBS and incubated for 15 min. Fixed cells were washed with DPBS, resuspended with DPBS+1%BSA, and stored at 4 °C for up to 7 days prior to analysis. For intracellular markers, fixed cells were treated with Permeabilization Buffer (eBioscience, Cat. # 00-8333-56). Cells were transferred to Falcon 96-well V bottom plates (Fisher Scientific, Cat. # 08772212) for immunostaining. Antibody incubations were performed on ice in the dark for 45 min in 100 ul/well of CAS-Block (Thermo Fisher Scientific, Cat. # 008120) supplemented with 1% BSA and 5% goat serum (plus 0.2% Triton for intracellular markers). Cells were then washed twice with 200 ul/well DPBS (cell surface markers) or Permeabilization Buffer (intracellular markers) before resuspended in 100 ul/well DPBS +1% BSA. Flow cytometry analysis was performed on the BD LSRFortessa with a High Throughput Sampler, with markers quantified in 9000 gated events on average. The antibodies used for detecting iPSC, endoderm, mesoderm, and ectoderm markers, as well as corresponding isotype controls, are listed in Table 2.