Foxp3 transcription factor staining buffer set

Immunophenotypic analysis was performed using one of the following instruments: BD FACSCanto II, BD LSRFortessa II, BD FACSymphony A5, Beckman Coulter Cytoflex S or Beckman Coulter Cytoflex LX. Antibodies used for immunophenotypic analysis are listed in Supplementary Data 7. Cells were incubated with a human (Miltenyi Biotec, #130-059-901, dilution 2:100) and mouse FcR blocking reagent (BD #553141, dilution 1:100) prior to staining with antibody cocktail. All steps were performed at 4 °C in PBS 2% FBS. Data were analyzed with FCS Express 6 and 7 (DeNovo Software). Staining for cell cycle analysis was performed by fixing and permeabilizing cells with the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set as per manufacturer’s indications, followed by 30 min incubation at +4^oC with human FcR blocking reagent (Miltenyi Biotec, #130-059-901, dilution 2:100) and overnight staining at +4^oC with Ki67-AF647 (BD #558615, dilution 2:100). Samples were acquired on the FACSymphony A5 with low flow rates after addition of Hoechst 33342. Staining for EdU incorporation was performed with the Click-iT™ Plus EdU Pacific Blue™ Flow Cytometry Assay Kit (TermoFisher Scientific #C10636) following the manufacturer’s indications.

For Th1 and Th2 differentiation OT-II TCR tg CD4⁺ T cells were cultured in IgG-coated 12 well plates for 3 days under Th1 or Th2 conditions. For Th1 skewing, cells were cultured in the presence of anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml), hIL-2 (30 units/ml), anti-IL-4 (2 µg/ml) and IL-12 (10 ng/ml). For Th2 differentiation, cells were cultured in the presence of anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml), hIL-2 (30 units/ml), anti-IFN-γ (10 µg/ml), anti-Il-12 (10 µg/ml), IL-4 (100 ng/ml). After 3 days, cells were stained for differentiation markers using anti-GATA3-APC, anti-Tbet-PE, anti-IL-4-PE, anti-IFN-γ-APC, anti-TNF-α-APC, anti-IL-2-APC, anti-IL-13-Alexa Flour (488) and either anti-CD4-FITC or anti-CD4-APC antibodies (pl. see Supplemental Table S2). For intracellular staining of cytokines cells were fixed and permeabilized using the eBioscience Intracellular Fixation & Permeabilization Buffer Set (Thermo Scientific #88-8824-00). Transcription factors (TBET, GATA3, MYC) were stained using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Miltenyi, #130-093-142). Cells were fixed for 45 min at room temperature, centrifuged and resuspended in permeabilization buffer supplemented with the corresponding staining antibodies.