Anti cd45 pacific blue

Cell counts were obtained with a NucleoCounter NC-250 (chemometec, Lillerød, Denmark) total cell and viability assays on A8 slides. For flow cytometry, after blocking with anti-CD16/32 antibody (1:100, clone 93, 14-0161-86; Thermo Fisher Scientific, Waltham, MA, USA), cells were stained with anti-CD45 pacific blue (1:100, clone 30-F11, MCD4528; Thermo Fisher Scientific), anti-CD11c APC (1:200, clone N418, 17-0114-82; Thermo Fisher Scientific), and anti-Ly6G Alexa488 (1:200, clone RB6-8C5, 53-5931-82; Thermo Fisher Scientific) antibodies. Acquisition was performed with an LSR II machine (BD Biosciences, Franklin Lakes, NJ, USA), with ≥5000 CD45⁺ cells collected/sample. FlowJo software v.10 (Tree Star, Ashland, OR, USA) was used for analysis. BAL macrophages and neutrophils were defined as CD45⁺CD11c⁺Ly6G⁻ and CD45⁺CD11c⁻Ly6G⁺, respectively, and their concentrations were calculated by multiplying the percentage observed by the total cell count.

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.

Using 0.2% II type collagenase (Sigma, USA), inflamed TA muscles were collected and digested for 40 min at the condition of 37 °C. In vivo, the single cell suspension obtained from muscle homogenate was blocked. In vitro, cultured cells were digested with Trypsin (Sigma, USA), resuspended in ice cold PBS to obtain the single cell suspension. The following fluorescent antibodies were used: anti-CD45-Pacific Blue, anti-F4/80-PE, anti-CD11b-PE, anti-MHC-II-eFluor 450, anti-Ly6C-FITC, anti-CX3CR1-APC, anti-CD206-eFluor 700, anti-Bcl3-FITC, anti-CD31-APC, anti-IL-10-FITC, rabbit anti-p-STAT3-FITC, the antibodies above were purchased from ThermoFisher and their dilution ratios were 1:100; Other antibodies involved anti-Vav1-FITC (1:100, Biorbyt, USA), anti-Rac1-GTP-FITC (1:100, Proteintech, USA), anti-Tunel-FITC (5:50, Yeason, China), anti-Annexin-V-APC (5:100, Sigma, USA), anti-CRT-Alexa Fluor 647 (1:100, Abcam, UK), anti-PKH67-Alexa Fluor 647 (1:250, Zeye, China), anti-CD36-Alexa Fluor 700 (1:100, eBioscience, USA), and anti-PPARγ-FITC (1:100, Abcam, UK). To analyze the labeled cells, FACSAria II cell sorter with FlowJo software (BD Biosciences, USA) were used.