Cd24 buv395

KiPS chemical resetting to naive at passage 6–7 were dissociated into single cells using TrypLE and passed through 50‐μm cell strainers (VWR). Conjugated antibodies and Fixable Viability Dye‐eF780 (eBioscience) were mixed with 50 μl of Brilliant stain buffer (BD Biosciences, 566349) and applied to 50 μl of cells (500,000 cells per reaction). Cells were incubated for 30 min at 4°C in the dark and washed with 2% foetal bovine serum (FBS) in PBS and centrifuged at 300 g for 3 min. Cells were resuspended in 2% FBS in PBS and analysed with a BD FACSAria Fusion for cell sorting. The following fluorescent conjugated antibodies and flow cytometer laser and filter settings were used: CD24‐BUV395 (BD Bioscience, 563818, 1.25 μl per test; detected using the 355‐nm laser with 379/28 filters), CD90.2‐APC‐Cy7 (BD Bioscience, 561641, 2.5 μl per test; 640‐nm laser with 780/60 filters), Fixable Viability Dye‐eF780 (ThermoFisher, 65‐0865‐14, 0.6 μl per test; 640‐nm laser with 780/60 filters) and SUSD2‐FITC (MACS Miltenyi Biotec, 130‐106‐327, 0.25 μl per test; 488‐nm laser with 530/30 filters).