Fixable viability dye ef780

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).

Immediately after cell purification, 1 × 10⁶ cells were stained with Fixable Viability Dye eF780 (eBioscience, San Diego, CA, USA) in 200 μL PBS in a 96‐well U‐bottom plate. The cells were pelleted and supernatant removed. Antibodies against surface antigens were added in 100 μL PBS and 1% FBS at the optimal concentrations determined by previous titration (Table 3) and incubated for 15 min at room temperature in the dark. Following two washes with PBS and 1% FBS, intranuclear FoxP3 staining was performed utilizing the FoxP3/Transcription Factor Staining Kit (eBioscience) per manufacturer's instructions including the recommended 15‐min incubation with mouse serum prior to intranuclear staining. Cells were resuspended in PBS and 1% FBS, transferred to FACS tubes, and analyzed within 6 hrs. Either isotype staining or Fluorescence Minus One (FMO) stains were performed. For isotype control staining, the appropriate antibody isotype or, if unavailable, a similar isotype coupled to the same fluorophore was used at the same concentration as the antibody. Histogram gating was performed using the isotype staining as a guide to set the gates. For FMO controls, all antibodies minus one (FoxP3) were stained.9 Analysis was performed on a BD SORP LSR II analytic flow cytometer with post‐acquisition analysis performed with flowjo (Treestar, Palo Alto, CA, USA).

Single cells were incubated with anti-mouse CD16/32 (93, TruStain fcX; BioLegend) to block Fc receptors. Staining was performed with Fixable Viability Dye eF780 or eF506 (eBioscience), to identify dead cells.The following fluorochrome-conjugated anti-mouse mAbs were used: CD3e-AF488 (145-2C11), CD4-PeCy7 (GK1.5), Foxp3-APC (FJK-16s), GATA3-PE (L50-823), CD45-APC-eFluor780 (30-F11), CD11c-BV605 (N418), Siglec-F-PE (E50-2440), CD11b-BUV737 (M1/70), MHCII-PeCy7 (M5/114.15.2), CD45.2-BUV395 (104), CD62L-FITC (MEL14), BrdU-AF647 (3D4), ST2-BV421 (U29-93), CD127-PeCy7 (A7R34), and Thy1.2-PerCP-eFluor710 (30-H12). Flow cytometric analysis was performed using a LSR Fortessa X-20 flow cytometer (BD Biosciences) and FlowJo software (Tree Star). Intracellular staining was performed using eBioscience Fixation Permeabilization buffers. BrdU staining was performed following the BD Biosciences manufacture’s protocol.