SVCs were resuspended in 500 mL of magnetic bead buffer (MBB) consisting of PBS without calcium and magnesium, 0.5% w/v bovine serum albumin (BSA), and 2 mM ethylenediaminetetraacetic acid (EDTA). The cell suspension was then filtered through a 30-mm pre-separation filter (Miltenyi, Bergisch Gladbach, Germany) following three filter washes to remove any large particles and debris.
The resulting cell suspension was then separated at 300 × g for 5 min at 4°C and resuspended in 90 mL of MBB along with 10 mL of anti-CD45 magnetic beads-conjugated antibody (Miltenyi) for each 10ˆ7 cell sample. The cell suspension was incubated for 15 min at 4°C, then diluted with 2 mL of MBB and centrifuged. The resulting cell pellet was resuspended in 500 mL of MBB, applied onto hydrated MS-columns (Miltenyi), washed three times with 500 mL of MBB, and collected with 1 mL of MBB through piston elution.
The resulting cell suspension was then separated at 300 × g for 5 min at 4°C and resuspended in 90 mL of MBB along with 10 mL of anti-CD45 magnetic beads-conjugated antibody (Miltenyi) for each 10ˆ7 cell sample. The cell suspension was incubated for 15 min at 4°C, then diluted with 2 mL of MBB and centrifuged. The resulting cell pellet was resuspended in 500 mL of MBB, applied onto hydrated MS-columns (Miltenyi), washed three times with 500 mL of MBB, and collected with 1 mL of MBB through piston elution.