Red blood cell lysis solution

C2C12 murine myoblasts were purchased from ATCC and grown and differentiated into myotubes as previously reported^{15 (link)}. Experiment was performed as previously described^{15 (link)}. Briefly, primary myoblasts were isolated from leg muscles of mice. Muscles were minced mechanically and digested with trypsin and collagenase D in F12 media for 1 h–3 h at 37 °C. Cells were resuspended with PBS and incubate 1 min at RT in Red Blood Cell Lysis Solution (Sigma, cat#R7757). Isolation of myoblasts was performed using magnetic beads from Miltenyl Biotec (MACS Satellite Cell Isolation Kit (cat#130-104-268) and anti-INTEGRIN-7 MicroBeads (cat#130-104-261)), where cells were incubated on ice for 15 min prior to selection on columns (LS columns; Miltenyi Biotec, cat#130-042-401). Primary myoblasts were cultured on gelatin-coated dishes (Sigma, cat#G9382) in the following media: 39% DMEM with glutamax, 39% F12 with glutamax, 20% fetal bovine serum (Wisent) and 2% UltroserG (Pall Life Sciences, cat#15950-017). Myoblast differentiation was induced, by changing media for 2% horse serum in DMEM/F12 (with glutamax) for 72 h. Quantifications of the fusion index were performed manually by counting the number of nuclei per fiber using the Volocity software (PerkinElmer Life and Analytical Sciences).

Spleens were harvested from immunized mice (n = 9), and single-cell suspensions were prepared by passing the organs through 70-μm-pore cell strainers into RPMI-10. Cells were pelleted by centrifugation (450 × g), resuspended in red blood cell lysis solution (Sigma), and then incubated at room temperature for 10 min. Cells were washed twice by being pelleted (450 × g) and then resuspended in RPMI-10, and then they were passed through a 40-μm-pore cell strainer (Falcon). Prior to treatment for T cell depletion, splenocytes prepared from 3 individual spleens were pooled, resulting in 3 groups of splenocytes. Each group was then split into 3 equal aliquots and individually processed for (i) CD4⁺ T cell depletion or (ii) CD8⁺ T cell depletion or (iii) were not depleted. CD4⁺ or CD8⁺ T cells were depleted by positive selection using magnetic antibody cell separation (MACS) CD4 (L3T4) MicroBeads and CD8a (LY-2) MicroBeads with LD separation columns (Miltenyi) per the manufacturer’s instructions. The undepleted samples (not treated with MicroBeads) were run on MACS LD separation columns for control purposes. The column eluates were pelleted by centrifugation (450 × g) and then resuspended in RPMI-10. Mouse IFN-γ ELISpot assays were conducted as described above.

Splenocytes were treated with red blood cell lysis solution (Sigma-Aldrich, Inc., St. Louis, MO, USA) at 4°C for 5 min, and washed twice with PBS containing 1% fetal bovine serum. Subsequently, splenocytes were incubated with FITC-conjugated hamster anti-mouse CD3e (CD3ε chain) monoclonal antibody (BD Biosciences, San Jose, CA, USA), followed by PE-conjugated rat anti-mouse CD45R/B220 monoclonal antibody (BD Biosciences). To measure the proportions of CD4+ and CD8+ T cells in splenocytes, cells were incubated with PE-conjugated rat anti-mouse CD4 (L3T4) monoclonal antibody (BD Biosciences) and FITC-conjugated rat anti-mouse CD8 monoclonal antibody (BD Biosciences) for 30 min at 4°C. Cells were washed twice with PBS, resuspended in 1 ml PBS, and analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

All fresh samples were processed as follows: For scRNA‐seq, tumor tissue samples were extracted with blades, single cell suspensions were acquired using a Tumor Dissociation Kit (130‐095‐929, Miltenyi Biotec) and a DNaseI (DN25‐100MG, Sigma Aldrich, St. Louis, MO, USA) digestion in a medium (RPMI1640 with 5% fetal bovine serum (FBS)) for 30 min at 37 °C. To remove cell aggregates or other large residual particles from the single‐cell suspension, the cell suspension was filtered through a 40‐um nylon mesh. Red Blood Cell Lysis Solution (10 × ) (Sigma‐Aldrich) and a Dead Cell Removal Kit (Miltenyi Biotec) were used to remove erythrocytes and dead cells, respectively. Library preparation was conducted using a 10 × chromium single‐cell kit following the manufacturer's protocol. Libraries were sequenced using the NovaSeq sequencing platform. The Cell Ranger software pipeline (version 6.0.1) was used to process 10 × raw genomic data. Cell Ranger was applied to demultiplex raw base call files into FASTQ files and for alignment, filtering, barcode counting, and unique molecular identifier (UMI) counting.