Cd25 alexafluor488

Spleen and MLN single cell suspensions (0.5–1 × 10⁶ cells/well) were incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block, BD Biosciences, San Jose, CA, USA) in PBS supplemented with 1% BSA and 5% FCS for 15 min on ice to block non-specific binding sites. Subsequently, cells were incubated for 30 min with the following antibodies: CD4-PerCP Cy5.5, CCR6-PE (both from Biolegend, San Diego, CA, USA) CD8a-PECy7, CD69-PE, CD25-Alexa Fluor 488, CD3-PerCP Cy5.5, CD27-PE, CD19-APC and B220-FITC (all from Thermo Fisher). For intracellular staining, cells were first fixated and permeabilized with Foxp3 Staining buffer set (Thermo Fisher) according to manufacturer’s protocol, followed by incubation with Foxp3-PECy7 (Thermo Fisher), RORγT-Alexa Fluor 647 (BD) or Tbet-eFluor 660 (Biolegend). Dead cells were excluded using Fixable Viability Dye eFluor^® 780 (Thermo Fisher). Stained cells were measured by FACS Canto II (BD Biosciences) and analyzed using Flowlogic software version 7 (Inivai Technologies, Mentone, VIC, Australia).

Antibodies for Western blotting were purchased from the following sources: Histone H2A (Cell Signaling Technology #3636); HMGB1 (Cell Signaling Technology #6893); β-Actin (Cell Signaling Technology #3700); Profilin-1 (Cell Signaling Technology #3237); Enolase-1 (Cell Signaling Technology #3810); Bip/GRP78 (Cell Signaling Technology #3177); Alpha-fodrin (EMD Millipore #MAB1622); MLKL (Cell Signaling Technology #14993); Phospho-MLKL Ser358 (Cell Signaling Technology #91689); GAPDH (ThermoFisher #AM4300). Antibodies for flow cytometry were purchased from the following sources: F4/80, PE-Cyanine5 (ThermoFisher #15-4801-82); CD11b APC-eFluor 780 (ThermoFisher #47-0112-82); CD11c Alexa Fluor 700 (ThermoFisher #56-0114-82); CD86 (B7-2) FITC (ThermoFisher #11-0862-82); CD40 Super Bright 436 (ThermoFisher #62-0401-82); MHC Class II I-Ab (ThermoFisher #17-5320-82); CD25 Alexa Fluor 488 (ThermoFisher #53-0251-82); CD69 Alexa Fluor 700 (BioLegend #104539).

MLN and spleen single-cell suspensions were prepared for flowcytometry analysis as previously described [61 (link)]. The cell suspensions were incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block; BD Biosciences, Franklin Lake, NJ, USA) in PBS + 1% BSA for 15 min on ice to block unspecific binding sites. Afterwards, cells were stained with the following surface markers CD4-PerCp-Cy5.5, CD69-PE-Cy7, CXCR3-PE, CD25-AlexaFluor488, CD25-PE, (all purchased from eBioscience, San Diego, CA, USA) or T1ST2-FITC (MD Bioproducts, St. Paul, MN, USA) for 30 min on ice. Fixable Viability dye eFluor 780 (eBioscience) was used to exclude non-viable cells. Next, the cells were fixed and permeabilised with the Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer’s protocol and then stained with the intracellular markers Foxp3-PE-Cy7, RORɣt-PE, IRF4-FITC, Tbet-eFluor660 or Gata3-eFluor660 (all purchased from eBioscience). Cells were measured on BD FACSCanto II flow cytometer, and results were analysed with FlowLogic software (Inivai Technologies, Mentone, Vic, Australia). The used gating strategy is shown in Figure S1.