Apc conjugated anti cd45 antibody

Mice were anesthetized and perfused with ice-cold PBS. Brains and SCs were dissected and after removal of meninges, and digested as described previously [25 (link)]. Cell suspensions were subjected to myelin removal using myelin removal beads II kit (cat. 130-096-733; Miltenyi Biotec) and cell pellets were suspended in 1 ml of staining buffer (PBS containing 2% bovine serum albumin and 0.09% sodium azide) and then subjected for mass cytometry (see experimental procedure in Additional file 1). The perfusion quality was evaluated as previously described [26 (link)–28 ]. Briefly, cells in the blood circulation were labeled by injecting the PE-conjugated anti-CD45 antibody (BioLegend) to the tail vein of mice (3 μg/mouse, in 150 μl of PBS). Thus, 97% of the immune cells in the circulation were labeled and following perfusion enabling us to identify the cells that originated from the blood and present in CNS compartment by their PE-conjugated anti-CD45 antibody labeling. To distinguish the CNS-resident immune cells from these originated from the blood, we stained the cells extracted from the CNS with an APC-conjugated anti-CD45 antibody (Biolegend). CNS compartment contained 4.2% immune cells, while only 0.5% blood-derived immune cells (Additional file 2).