Mouse mdscs isolation kit

For mouse BMDCs, femurs were obtained from 4–6-wk-old C57BL/6 mice, and bone marrow was flushed aseptically with RPMI medium using a syringe fitted with a 27-gauge needle. Mouse CD8⁺ T cells were isolated from mouse spleen using a CD8⁺ isolation kit (Miltenyi Biotec; 130-104-075). MDSCs were isolated from spleens of tumor-bearing mice using a mouse MDSCs isolation kit (Miltenyi Biotec; 130-094-538). Cell purity was checked by flow cytometric analysis using anti-CD11b and Gr-1 antibodies (>95%), and cell viability was checked by Trypan blue dye exclusion. The resulting cells were cultured using RPMI medium supplemented with 10% FBS at 37°C, 5% CO₂, and used for further tumor-induced MDSC conversion, MDSC expansion, and MDSC function assays.
For the mouse tumor-induced MDSC conversion assay, BMDCs were derived from healthy 4–6-wk-old C57BL/6 mice and cocultured with PANC02-vector/PANC02-EHF. After 6-d coculture, cells were collected and stained for CD45, CD11b, and Gr-1. For mouse MDSC expansion assays, isolated MDSCs were stained with CFSE. Then, labeled MDSCs were cocultured with PANC02-vector/PANC02-EHF at 5:1 for 72 h. Proliferation was assessed by measuring CFSE dilution by flow cytometry.

MDSCs were isolated from tumor tissue collected from tumor-bearing mice using a mouse MDSCs Isolation Kit (Miltenyi Biotec, America) according to the manufacturer’s instruction. For coculture assays, prior to the experiment, the plates (6-well) were pre-coated with 1mL of PBS containing 5 μg/mL anti-CD3 antibody and incubated overnight at 4°C. The extracted CD8⁺ T lymphocytes were counted, centrifuged, and the supernatant was discarded. The T lymphocytes were then resuspended in 2 mL of RPIM 1640 complete culture medium containing 5 μg/mL CFSE and incubated for 20 min at 37°C. The incubation was terminated with 10 mL of complete culture medium, followed by centrifugation and discarding of the supernatant. Each well was then seeded with 1 × 10⁶ CFSE-labeled CD8⁺ T lymphocytes. In the wells containing T lymphocytes with activators, 0.5 × 10⁶ MDSCs, 2 × 10⁶ MDSCs, and 5 × 10⁶ MDSCs were added respectively. The culture medium was adjusted to 2.5 mL, and 5 μg/mL anti-CD28 was added. The plates were then incubated at 37°C with 5% CO2 for four days. After four days, the cells in the culture plate were collected and analyzed for CFSE level.