Anti thy1.1 antibody

SCs were obtained from the sciatic nerve of Sprague-Dawley (SD) rats 1 d after birth, and primary cultured in vitro. Rats were acquired from the Experimental Animal Center of Nantong University [license No. SCXK (Su) 2014-0001 and SYXK (Su) 2012-0031, No. 20190225-004]. The culture method used has been previously described (14 (link)). In brief, cells were cultured in DMEM medium (Corning, USA) supplemented with 10% of fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), then anti-Thy1.1 antibody (Sigma, St. Louis, MO, USA) and rabbit complement (Invitrogen, Carlsbad, CA, USA) were added to remove fibroblasts. Purified SCs were identified by immunostaining and plated in 96- or 6-well plates, with the culture medium replaced by DMEM medium with 10% of FBS containing 0.5 µg/mL ABPPk or 0.1 µg/mL NGF, respectively. Multiple time points (15 min, 0.5, 1, 2, 3, 6, 12, and 24 h) were selected for detection and analysis.

Animal protocols were reviewed and approved by the Animal Ethics Committee of Nantong University, China (license No. 2014-0001), and the experimental protocol was in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication, No. 80-23). Precautions were taken to minimize suffering and the number of animals used in each experiment.
Primary cultures of Schwann cells were prepared as previously described (Brockes et al., 1981; Chen et al., 2012). In brief, Schwann cells were harvested from sciatic nerves of 1–3-day-old Sprague-Dawley rats (Experimental Animal Center of Nantong University, Nantong, Jiangsu Province, China, SPF level, no gender requirement). To inhibit fast proliferation of fibroblasts, the Schwann cell purification medium was replaced (Table 1) for 1 day, followed by a 1-day recovery period in growth factor-free medium, then refreshed with Schwann cell growth medium. About 7 days later, when the Schwann cell cultures reached confluence, complement-mediated immune cytolysis was performed to eliminate fibroblasts by incubating the cells in 4 µg/mL of anti-Thy-1.1 antibody (Sigma-Aldrich, St. Louis, MO, USA) for 2 hours on ice and then with 1 mL of rabbit complement (Gibco, Carlsbad, CA, USA) for 1 hour at 37°C. Afterwards, > 98% pure Schwann cells were obtained and the purity was determined by immunocytochemistry.

Neonatal Sprague-Dawley rats were anesthetized and then killed by cervical dislocation. Sciatic nerve stumps were collected from which primary Schwann cells were isolated. To remove contaminating fibroblasts, isolated cells were treated with an anti-Thy1.1 antibody (Sigma-Aldrich, St. Louis, MO, USA) and rabbit complement (Sigma-Aldrich). Cells were immunostained for a Schwann cell marker using a rabbit anti-S100 antibody (Dako, Carpinteria, CA, USA) and a secondary goat anti-rabbit antibody (Abcam, Cambridge, MA, USA) to determine the purity of the collected Schwann cells. Purified Schwann cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco) in a humidified 5% CO₂ incubator at 37°C. Cultured Schwann cells were passaged less than three times before use.

Schwann cells were harvested as previously described (Liu et al., 2019). Briefly, sciatic nerves were isolated from postnatal wild-type rats and green fluorescent protein-transgenic rats and digested with 3 mg/mL collagenase for 30 minutes and 0.125% trypsin for 10 minutes at 37°C. Isolated Schwann cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin for 24 hours. Cytosine arabinoside (10 µM; Sigma, St Louis, MO, USA) was added to the cell culture medium for 24 hours, and then 2 µM forskolin (Sigma) and 50 ng/mL heregulin (Sigma) were supplied to the culture media. Cells were then purified by the addition of anti-Thy1.1 antibody (1:1000; Sigma) and rabbit complement protein (1:3; Sigma) to remove contaminating fibroblasts.