Cd140a apc

Cells were stained with the following antibodies for analysis CD3-FITC (35–0031, Tonbo Bioscience), CD8-BV711 (100748, BioLegend), CD4-BV650 (100546, BioLegend), CD45-BUV395 (565967, BD Biosciences), CD90-BV785 (105331, BioLegend), CD11c-APC (20–0114, Tonbo Biosciences), MHC-II-Pac Blue (107620, BioLegend), CD103-PercPCy5.5 (121416, BioLegend), CD11b-A700 (557960, BD PharMingen), EpCAM-PercPe710 (46–5791-82, eBioscience), Ly51-PE (12–5891-83, eBioscience), CD31-PECy7 (25–0311-82, eBioscience), CD140a-APC (135907, BioLegend), UEA1-FITC (FL-1061, Vector Laboratories), TCRbeta-PECy7 (109222, BioLegend), CD62L-APC-Cy7 (104427, BioLegend), CD44-Alexa Fluor RTM700 (56–0441-82, BioLegend), CD25-PercP-Cy5.5 (102030, BioLegend). Annexin V staining (640906, BioLegend) was performed in Annexin V binding buffer (422201, BioLegend). Flow cytometric analysis was performed on an LSRFortessa X50 (BD Biosciences) and cells were sorted on an Aria II (BD Biosciences) using FACSDiva (BD Biosciences) or FlowJo (Treestar Software).

The tibialis anterior, gastrocnemius, and quadricep muscles were isolated from each mouse and placed in a 1.5 mL tube containing 500 μL of phenol-free, serum-free DMEM (GIBCO). Tissues were then minced until disintegrated. Minced tissues were further digested (30 min, 37°C) in DMEM containing collagenase VIII (Sigma Aldrich) and dispase (Thermo Fisher Scientific). Tissue homogenate was filtered into a new 50 mL conical using a 70 μM filter and washed with phenol-free DMEM (GIBCO) containing 2% FBS and EDTA (staining buffer). Cells were then centrifuged (10 min, 1500 rpm, 4°C) and resuspended in staining buffer, twice. Buffer was aspirated and pellet was resuspended in 250 μL of staining buffer containing the following antibodies (Biolegend): CD45 BV605 (1:100), CD31 PE-Cy7 (1:100), CD140a APC (1:50), Sea1 PerCP-Cy5.5 (1:50), and Vcam1 PE (1:100). Cells were incubated for 20 min then washed twice (5 min, 1500 rpm, 4°C) and resuspended in 200 μL of staining buffer. Stained cells were then sorted using a FACS Aria II (Becton Dickinson). Cells were sorted twice to ensure purity.