Cd3 efluor450

For children a 100 μl volume of whole blood for each donor was stained with antibodies to the following surface markers: CD3 PE, CD4 PErCP, (BD Biosciences), CD8 efluor450 and CD27 APCalexafluor750 (Ebioscience) for 30 min at 4°C. Red blood cells were then lysed using 1:10 FACS Lysing Solution (BD Biosciences) and incubated for 10 min at room temperature. Cells were then washed twice in FACS buffer (PBS, 5% BSA, 5% EDTA) and re-suspended in Cytofix (BD, Biosciences). Samples were acquired on a Cyan ADP flow cytometer using Summit software (Beckman Coulter) at MRC Gambia.
Lymphocyte subsets from AIM patients were identified by staining with antibodies specific to: CD19 FITC, CD4 PE (Biolegend), CD27 APC elfluor 780, CD3 efluor 450 (eBioscience) and CD8 qDot 655 (Invitrogen). Samples were stained for 30 min on ice, washed and analysed immediately on an LSR-II flow cytometer (BD Biosciences). Data was analysed using Flow-Jo software (Treestar Inc).

Two days prior to mRNA immunization, 2 × 10⁶ OT-I or OT-II cells were purified and labeled with 5 µM of CFSE (Invitrogen, Merelbeke, Belgium) and subsequently adoptively transferred via i.v. injection into mice that had been s.c. inoculated with B16 cells. Four days after the mRNA treatment, draining lymph nodes were isolated and OT-I or OT-II cell division was analyzed by flow cytometry. Cells were stained with anti-CD16/CD32 (500× dilution) (BD Biosciences) to block Fc receptors followed by staining with Fixable Viability Dye (1000× dilution) (BD Biosciences), CD8 PE-Cy7 (200× dilution) (eBiosciences), CD3 efluor450 (200× dilution) (eBioscience), anti-CD19 allophycocyanin (APC; 200× dilution) (BD Biosciences), and MHC-I dextramer H-2 Kb/SINFEKL-PE (500× dilution) (Immundex). The experiments were performed on a triple-laser (B-V-R) LSR-II (Becton Dickinson, San Jose, CA, USA), and data were analyzed using the FlowJo software (Treestar, OR). Single cells were gated based on FSC and SSC. Living cells were selected and T cells gated for CD3⁺ CD19⁻ T cells. Within the CD8⁺ T cells or CD4⁺ T cells, OVA-specificity was gated by labeling with MHC-I SINFEKL-PE dextramer. See Supplementary Fig. 4 for the gating strategy.

Hind limb muscles from C57/BL6 wild type mice (Charles River Laboratories) were minced and digested in PBS (Sigma) containing 0.1% BSA, 300 µg/ml Collagenase A (Roche), 0.24 U/ml Dispase I (Roche), 2 μg/ml DNase I (Roche), 50 μM CaCl2 and 1 mM MgCl2 for 60 min at 37 °C under constant agitation. For fluorescence-activated cell sorting, digested muscle cells were stained with primary antibodies (1:50) CD3-eFluor450 (eBioscience), CD45-eFluor450 (eBioscience), Ter119-eFluor450 (eBioscience), Sca-1-FITC (BD Pharmingen), and α7integrin-APC (AbLab) for 30 min at RT. Cells were finally washed and resuspended in Running Buffer (PBS, 0.1% Sodium Azide, 0.2% FBS). Flow cytometry analysis and cell sorting were performed on a DAKO-Cytomation MoFlo High Speed Sorter.
Muscle satellite cells (SCs) were isolated as Ter119−/CD45−/CD31−/α7-integrin+/Sca-1− cells. Fibro-adipogenic progenitors (FAPs cells) were isolated as Ter119−/CD45−/CD31−/α7-integrin−/Sca-1+ cells, as previously described^{44 (link)}.

The following anti-mouse mAbs were used for surface staining during flow cytometry: CD3-eFluor 450, CD8-PerCP-Cy5.5, CD11b-AlexaFluor 700, CD11b-eFluor 450, CD14-FITC, CD25-APC, CD25-eFluor 450, CD45.1-APC, Ly6C-APC-eFluor 780, I-A/E-FITC (eBioscience, San Diego, CA, USA), CD45.1-eFluor 450, CD45R-Horizont V500, CD3-Horizont V500, CD4-Horizont V500, CD4-PerCP, CD8-Horizont V500, Ly6G-AlexaFluor 700, Siglec F-PE (BD Biosciences, San Jose, California, USA) and TLR4-APC (BioLegend, San Diego, Ca, USA). For intracellular staining, the following anti-mouse mAbs were used: TNFα-PE, Foxp3- and IFNγ-PE (eBioscience; San Diego, California, USA). Fc-block (anti-CD16/CD32 mAbs; eBioscience, San Diego, California, USA) was used both in surface and intracellular staining. For ELISA, matched anti-mouse capture mAb and biotinylated detection mAb against TNF-α, IFN-γ, IL-1β, IL-12 and IL-6 were used (R&D System, Minneapolis, Minnesota, USA). Blocking anti-mouse CD25 (PC61.5), CD4 (GK1.5) and anti-IFN-γ (XMG1.2) mAbs, as well as anti-mIL-2 mAbs for preparing IL-2 complexes (S4B6, JES6-1A12), were obtained from BioXcell (Lebanon, New Hampshire, USA).

CT26 tumors were digested using a gentleMACS dissociator and a murine tumor dissociation kit (Miltenyi Biotec). Absolute viable cell counts were determined by propidium iodide staining and analysed on the MACSQuant analyzer. Cells from CT26 tumors and splenocytes were stained with fixable LIVE/DEAD blue (Life Technologies) and incubated with anti-mouse CD16/CD32 (eBioscience) prior to addition of anti-mouse: CD8-Pe-Cy7 (clone 53–6.7); CD3-eFluor 450 (clone 17A2); CD11c-PE (clone N418); CD86-FITC (clone GL1); PDCA1-APC (clone 129c) (eBioscience); PD-L1-BV421 (clone 10F.9G2); I-A/I-E (MHCII) (clone M5/114.15.2); B220-BV605 (clone RA3-6B2) (Biolegend); CD45-BV785 (clone 30F11); CD4-BUV395 (clone GK1.5); CD11b-BUV395 (clone M1/70); Ly6G-APC-Cy7 (clone 1A8); Ly6C-PerCP-Cy5.5 (clone AL-21) (BD Biosciences). For intracellular staining, cells were permeabilized using Foxp3 / Transcription Factor Staining Buffer Set (eBioscience) and incubated with anti-mouse Foxp3-PE (clone FJK-16S) and Ki67-eFluor 660 (clone SolA15) (eBioscience). Stained cells were fixed in 3.7% formaldehyde and analyzed using a BD LSRFortessa (BD Bioscience). Data analysis was performed using FlowJo (FlowJo LLC). 8 mice per treatment group were included in all flow cytometry analyses.

Strains (5 × 10⁸ CFU/day/mice) were administered by intragastric gavage to WT conventional BALB/c mice for 5 days. Colon samples were removed at sacrifice and stored in RNAlater® storage solution (Ambion, Life Technologies, Foster City, CA, USA) at − 80 °C until qRT-PCR analysis. MLN and intestine were harvested and immediately processed for flow cytometry. Cell suspensions of MLN and intestine (3–5 × 10⁶ cells, in RPMI1640 supplemented by 10% FCS, 2 mM l-glutamine, 2 mM HEPES, 40 mg/ml gentamycine) were stimulated using the Leukocyte Activation Cocktail containing BD GolgiPlug (BD Biosciences) (1 μl/ml of cell suspension) for 5 h. Cells were stained by mAbs anti-mouse CD11c eFluor450, CD11b eFluor 450, B220 eFluor 450, CD3 eFluor 450, CD117 Alexa Fluor 700, NK1.1 PerCP-Cy5.5, NKp46 FITC (provided by eBioscience), CD4 APC-H7, CD90.2 BV 500; CD45RB BV 605, MHCII BV 650 (provided by BD Bioscience, san Jose, CA, USA). Subsequently, cells were permeabilized and fixed using the Transcription Factor Buffer Set (BD Bioscience), and intracellular staining was performed using mAbs anti-IL-17A eFluor450, anti-FOXP3 PE-Cy7, anti-IL-22 PE and RORgt APC (eBioscience). Flow cytometry data were analyzed using software FlowJo. Gating strategy and representative dot plots for control and BIO5768 treated mice are presented in Figs. S1 and S2.