Anti mhcii efluor450

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.

Tumors were dissociated as for cell inoculation and subjected to flow cytometry (FC) analysis, gating on live cells lacking staining with Live/Dead Aqua (ThermoFischer). All antibody staining was preceded by FcγR block on ice (eBioscience/Fisher). Extracellular antibodies were then added and samples were incubated on ice. Intracellular staining was accomplished after surface staining using the FoxP3 staining kit (eBioscience). To evaluate myeloid subsets, total cells were stained with anti-CD11b-FITC, anti-CD45-BV650, anti-Ly6C-AF700, anti-MR-PE-Cy7, anti-CD11c-PE/Dazzle594, and anti-Ly6G-BV605 (BioLegend); anti-MHCII-eFluor450 (eBioscience); anti-CD86-PE (Miltenyi); and anti-F4/80-APC (BioRad). T cells were enumerated by staining with anti-CD45-AF700, anti-CD3-AF488, anti-CD4-PE, anti-CD8-BV655, and anti-CD69-APC-Cy7 (BioLegend), followed by intracellular stain with anti-IFNγ-APC (BioLegend). To evaluate Tregs, total cells were stained with anti-CD3-AF488, anti-CD4-BV605, and anti-CD25-PerCP-Cy5.5 (BioLegend), and then stained intracellularly with anti-FoxP3-PE (BD Pharmingen).