30 mm pre separation filter

Mouse cerebella were dissected in ice-cold PBS. Tissues from four animals were pooled and then cut into pieces smaller than 1 mm in size. These pieces were then transferred to ice-cold MACS Tissue Storage Solution (Miltenyi Biotec). For cell dissociation, the storage solution was replaced with RPMI 1640 Medium (Thermo Fisher Scientific). The tissue was pelleted and digested with 500 ml of pre-warmed Accumax (Innovative Cell Technologies) in a 1.5-ml tube at 37°C for 5 min. After digestion, the tissue pieces were dissociated by gentle trituration with a wide-bore pipet tip. The cell suspension was then filtered through a 30-mm pre-separation filter (Miltenyi) following three filter washes to remove any large particles and debris. Any remaining cell clumps unable to pass through the filter were subjected to another round of digestion and filtering to maximize the yield of single cells. The cell suspension was collected into 1.5 ml of ice-cold Resuspension Buffer (Lebovitz L15 medium with 2% FBS, 25 mM HEPES, 2 mM EDTA). After dissociation, cells were stained with Trypan blue, counted, and cryopreserved in 90% FBS/10% DMSO freezing medium. Single-cell suspensions with more than 85% viability were then used to generate single-cell cDNA libraries.

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.