Anti ly6c fitc

Cells isolated from tumor tissues were blocked with purified CD16/32 antibody for 10 min and then stained for MDSCs using anti-CD11b APC, anti-CD45 PE C-Y7, anti-Ly6G PE, and anti-Ly6C FITC antibodies (eBioscience, San Diego, CA) for 30 min on ice. For Tregs staining, the cells were first stained for extracellular Treg markers (CD4 and CD25) with anti-mouse CD4-FITC,- and anti-mouse CD25-PE for 30 min. The cells were then fixed using the FOXP3/Transcription factor staining buffer set (eBioscience cat.# 00–5523) according to the manufacturer’s protocol. The cells were subsequently washed and stained with DAPI for 10 min before analysis by flow cytometry and Flowjo software V10.

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.