Schwann cell isolation Sample Clauses

Schwann cell isolation. Sciatic nerve fragments with a length of approximately 35mm were taken bilater- ally from deeply anesthetized (0.5 ml of a combination of ketamine (50 mg/kg), xylazine (2.6 mg/kg) and acepromazine (0.5 mg/kg)) adult Xxxxxxx 344 rats (aver- age weight 160-180 g). The sciatic nerves were washed with ice-cold Hank’s bal- anced salt solution (HBSS, PAA Labora- tories, Austria) and the epineurium was stripped off with a fine forceps. Each sci- atic nerve fragment weighed on average 29.6 mg ± 1.7. Nerves were cut into 1mm pieces and plated on collagen type-I (5 mg/cm2, Pan Biotech, Germany) coated culture dishes in standard medium, which consists of Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal calf serum (FCS; Pan Biotech, Ger- many) and Penicillin/Streptomycin (100 U of penicillin, 100 mg/ml Streptomy- cin; Pan Biotech, Germany). The culture dishes with the sciatic nerve pieces were kept in a cell culture incubator at 37°C in a humidified atmosphere containing 5% CO2. After 7 days in culture, the sciatic nerve fragments were transferred into 24 well plates containing 500 µl HBSS with 3% trypsin, 1% collagenase and 1% hy- aluronidase (all Sigma, Germany) per well. After two hours of incubation at 37°C, tis- sue fragments were dissociated by tritura- tion through a glass Pasteur-pipette and a 20-gauge sterile needle. The suspension was centrifuged at 120 x g for 10 min at room temperature. After resuspension in standard medium, trituration through a 22-gauge hypodermic needle followed. The resulting cell suspension was cultured in standard medium for two days on poly- l-ornithin/laminin (P-Orn/Lam; both Sig- ma, Germany) culture flasks, which were coated as follows: flasks were incubated with 20mg/cm2 poly-l-ornithin in distilled H2O for 2 hours at 37°C, rinsed with dis- tilled H2O and incubated with 0.4 mg/cm2 laminin in PBS for 2 hours at 37°C. After 2 days in culture, the number of viable cells total before MACS and flow-cytom- etry/immunocytochemistry analysis was determined by counting the Trypan blue excluding cells using a hemocytometer.

Schwann cell isolation. After obtaining sciatic nerve biopsies from adult rats, nerve fragments were kept in