Manual macs system

Splenocytes were isolated from 3-4 week-old SKG mice after erythrocyte lysis using ACK Lysing Buffer. CD4⁺ T cells were sorted from single cell suspensions of the splenocytes by biotinylated mAb against CD4, streptavidin-coated magnetic beads and a manual MACS system (Miltenyi Biotec) according to the manufacturer’s protocol.

The CD4 + T cells were isolated from single-cell suspensions prepared from the splenocytes of unimmunized SKG mice, using a biotinylated mAb against CD4, streptavidin-coated magnetic beads, and a manual MACS system (all from Miltenyi Biotec), according to the manufacturer’s protocol. The CD4 + T cells were cultured for 5 days in culture medium at 37 °C under Th17 conditions: pre-coated 10 ng/ml anti-CD3, pre-coated 5 ng/ml anti-CD28, 10 ng/ml human IL-2, 10 ng/ml IL-6, 2.5 ng/ml TGF-β, 2.5 μg/ml anti-IFN-γ, and 2.5 μg/ml anti-IL-4. BrPA at 0, 10, or 20 μM was added for the last 3 days.

To isolate G-MDSCs from the single-cell suspensions of cultured BM cells from Balb/c mice, we used a biotinylated mAb against Ly6G and streptavidin-coated magnetic beads (Miltenyi Biotec) according to the manufacturer’s protocol. To isolate CD4⁺ T cells from single-cell suspensions prepared from Balb/c splenocytes, we used a biotinylated mAb against CD4, streptavidin-coated magnetic beads, and a manual MACS system (all from Miltenyi Biotec) according to the manufacturer’s protocol.