Anti t bet bv421

HSF was provided by Hangzhou KSBIO Science and Technology (BinJiang, Zhejiang, China). Quality control was performed following published methods (Fu et al., 2011 (link)).
The anti-mouse antibodies for fluorescence-activated cell sorting (FACS) were as follows: anti-CD45 FITC, anti-CD8 APC-H7, anti-Eomes PE-Cy7, anti-T-bet BV421, anti-PD-1 BV605, anti-CD4 PE, anti-CD44 BV510, and anti-CD62L PE-Cy7 from BD Biosciences.

Damaged TA muscles were collected and minced and then gently digested with 0.2% IIcollagenase at 37 °C for 45 min twice. Total cells isolated from muscle homogenate were re-suspended in fluorescence-activated cell sorting buffer (phosphate buffer solution, 0.5% bovine serum albumin, 2 mM EDTA) to obtain a single-cell suspension. After Fc receptor blocking with anti-CD16/CD32 (Biolegend, USA), cells were incubated with anti-CD45-Pacific Blue, anti-F4/80-PE, anti-Ly-6C-FITC, anti-CD11b-PE, anti-CD3ε-APC, anti-CD4-FITC, anti-IFN-γ-PE, anti-IL-4-APC, anti-IL-17α-PE-Cy7, anti-CD25-PE, anti-Foxp3-APC (1:100, ThermoFisher, USA), anti-T-bet-BV421(1:100, BD Biosciences, USA), anti-CTLA-4-APC (1:100, eBioscience, California, USA), anti-MHC-II-eFluor 450 (1:100, eBioscience, California, USA), and anti-CX3CR1-APC (1:100, Bioss, China). Labeled cells were analyzed with a FACSAria II cell sorter (BD Biosciences, USA) and FlowJo software. For cell sorting, mice were sacrificed on day 3 and 6 after CTX-myoinjury. Muscle samples were collected, minced, and incubated with anti-CD45-Pacific Blue and anti-F4/80-PE on day 3, or incubated with anti-CD3ε-APC and anti-CD4-FITC on day 6. CD45⁺ F4/80⁺ cells, or CD3ε⁺CD4⁺ cells were sorted by MoFlo XDP, for further RNA preparation and qRT-PCR analysis.