As soon as all ganglion or spinal cord pieces were dissociated (DRG, SG: ∼1.5-2h; Spinal Cord: 45-60min), the cell suspensions were filtered using a 40μm cell strainer (FALCON) and collected in a 15ml plastic tube. The digestion solution was diluted with 3ml aCSF and centrifuged at 100 g for 4min at 4°C. The supernatant was removed and the pellet resuspended in 0.5ml aSCF and 0.5ml complete Neurobasal medium (Neurobasal-A supplemented with L-Glutamine, B27 (all GIBCO) and Penicillin/Steptamycin (Sigma)). The cell suspension was carefully transferred with a Pasteur pipette and layered on top of an Optiprep gradient: 90μl (DRG) or 80μl (SG) Optiprep Density Solution (Sigma) in 455μl aCSF and 455μl complete Neurobasal; and for spinal cord 170μl of Optiprep in 915μl aCSF and 915μl complete Neurobasal. The gradient was centrifuged at 100 g for 10min at 4°C, the supernatant removed until only 100μl remained and 10μl DNaseI added to avoid cell clumping.
