Attune nxt

The following mAbs were used: anti-CD3 (17A2 and 145-2C11), anti-TCRβ (H57-597), anti-CD8 (53-6.7), anti-CD49b (DX5), anti-phospho-S6 (Ser-235/Ser-236), and anti-IL-18Rα (P3TUNYA) from eBioscience; anti-CD19 (1D3), anti-CD4 (RM4-5), anti-F4/80 (T45-2342), anti-NK1.1 (PK136), anti-Ki-67 (B56), and anti-BrdU (3D4) from BD Biosciences; anti-CD71 (RT7217), anti-CD98 (RL388) from Biolegend; and Live/Dead Fixable Yellow Dead Cell Stain from Invitrogen. Intracellular staining of Ki-67 was carried out using a Foxp3 staining kit (eBioscience). Mitochondrial mass was measured using nonylacridine orange (NAO) (Thermo Fisher Scientific). Cells were acquired using BD LSRFortessa or Thermofisher Attune NxT and analyzed using Kaluza 1.3 Analysis software (Beckman Coulter) or FlowJo (V10).

Surface staining for flow cytometry was done in column buffer (2 mM EDTA [Millipore], 10 mM HEPES [GE Healthcare Life Sciences Life Sciences], 5% [v/v] fetal bovine serum in PBS without calcium and magnesium) for 20 min at 4°C. After staining, cells were washed twice with column buffer, fixed overnight in a 1:1 fix solution of column buffer: 4% paraformaldehyde, and washed prior to running flow cytometry. For intracellular cytokine staining, cells were stimulated for 8–12 hr with a Cell Stimulation Cocktail plus protein transport inhibitors (eBiosciences) at 4°C. Intracellular staining was done after fixing and permeabilizing cells using Perm/Fix buffer (eBiosciences) overnight at 4°C or for 1 hr at room temperature. Cells were then washed and stained in a Perm/Buffer wash solution (eBiosciences) for 30 min at 4°C. Cells were then washed twice with the Perm/Buffer wash before being resuspended in column buffer. Flow cytometry data for conventional mouse experiments were collected with a BD LSRII-561 and analyzed with FlowJo (Version 10) software. Flow cytometry data for the germ-free experiment was collected on an Invitrogen Attune NxT or CytoFLEX (Beckman Coulter) machine. See Table 1 for antibodies used in this study. Single stain and Fluorescence Minus One controls were used for gating.

Cells from animals were stained with fluorescently conjugated antibodies: CD69-Brilliant Violet 510 (clone FN50) or CD14-Brilliant Violet 510 (clone M5E2), CD3-Pacific Blue (clone Hit3a), CD8a-FITC (clone Hit8a), CD4-PE (clone RPA-T4), CD19-PE-Cy5 (clone SJ25C1), CD45-APC (clone 2D1), CD56-PE-Cy7 (clone MEM-188) (all from Biolegend) and Ghost Dye Red 780 (Tonbo Biosciences). All flow cytometry samples were run using a MACSQuant Analyzer 10 flow cytometer (Miltenyi) or Attune NxT. (Beckman Coulter). All data was analyzed using FlowJo v.10 (TreeStart, Inc) (Supplementary Fig. 10d).