Percp cy5.5 cd45

Single-cell suspensions of the tumors were prepared as using the method described above. The cells were enriched using the CD45(TIL) Microbead Mouse Kit (Cat. No.: 130-110-618, Miltenyi Biotec, Lerden, the Netherlands) according to the magnetic-activated cell sorting (MACS) protocol and stained with the antibodies, Ghost Dye™ Violet 510 Viability Dye (Cell Signaling Technology) and Percp-Cy5.5-CD45 (Clone 30-F11) for FACS sorting. Approximately 1×10⁵ CD45⁺ cells/sample were sorted using a BD Aria II instrument. Based on the FACS analysis, single cells were sorted into flow tubes, and the cell viability was tested by determining the AOPI to ensure sufficient cell quality. Then, the cell suspension, which contained 300-600 living cells per microliter, as determined using CountStar, was loaded onto the chromium single-cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s instructions. Single-cell transcriptome amplification was performed with an S1000™ Touch Thermal Cycler (Bio-Rad) at 53°C for 45 min, followed by incubation at 85°C for 5 min, and holding at 4°C. The cDNA templates were generated and then amplified, and the quality was assessed using an Agilent 4200 instrument (performed by CapitalBio Technology, Beijing).

The procedure described above was used to make single-cell suspensions of tumor cells from the left tumors. The cells were enriched for FACS sorting using the CD45 (TIL) Microbead Mouse Kit (Catalog No. 130-110-618, Miltenyi Biotec, Leiden, the Netherlands) and labeled with the antibodies Ghost DyeTM Violet 510 Viability Dye (Cell Signaling Technology) and Percp-Cy5.5-CD45 (Clone 30-F11). A BD Aria II device was used to sort about 5 × 10⁵ CD45⁺ cells per sample. Single cells were sorted into flow tubes based on the FACS analysis, and cell viability was determined by calculating the AOPI to guarantee adequate cell quality. The cell suspension was then put onto the chromium single-cell controller (10X Genomics) to form single-cell gel beads in the emulsion according to the manufacturer’s directions, with 300–600 viable cells per microliter as measured by CountStar. An S1000TM Touch Thermal Cycler (Bio-Rad) was used to perform single-cell transcriptome amplification at 53 °C for 45 min, followed by incubation at 85 °C for 5 min and storage at 4 °C. The quality of the cDNA templates was tested using Agilent 4200 equipment after they were produced and amplified (performed by CapitalBio Technology, Beijing).