Ionomycin phorbol 12 myristate 13 acetate brefeldin a

Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5
min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS
buffer (1% BSA, 0.05% NaN₃, in PBS) or full RPMI 1640. Cells in FACS buffer
were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with:
anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all
BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend;
20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then
anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated
±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h,
37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8
(20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and
anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or
Ly6C⁺ cells, whole blood (ethylenediaminetetraacetic acid) was stained with
anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend),
anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing,
and analysis by flow cytometry (Accuri C6).

Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5 min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS buffer (1% BSA, 0.05% NaN₃, in PBS) or full RPMI 1640. Cells in FACS buffer were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with: anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend; 20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated ±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h, 37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8 (20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or Ly6C⁺ cells, whole blood (ethylenediaminetetraacetic acid) was stained with anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend), anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing, and analysis by flow cytometry (Accuri C6).