Collagenase 4

Lamina propria mononuclear cells (LPMCs) were isolated as previously described with minor modifications (31 (link)). Briefly, colons were cut longitudinally, washed in Hank’s balanced salt solution (HBSS) supplemented with 1% fetal bovine serum (FBS). Tissues were cut into 0.5-cm² pieces and incubated at 37°C for 20 min in HBSS containing 3 mM ethylenediaminetetraacetic acid and 1mM dithiothreitol to remove epithelial cell. After the incubation, epithelial cells in supernatant were discarded by passing through a 100-µm cell strainer. Then, the remaining pieces was minced and incubated with 1× minimum essential medium–α containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS, and supplemented with 1 mg/ml collagenase IV (Biosharp, China), 1 mg/ml dispase II (Roche, Basel, Swiss), and 0.5 mg/ml deoxyribonuclease I (Roche, Basel, Swiss) for 60 min in a shaking incubator at 37°C. The resulting cell suspension was filtered through a 70-μm cell strainer (BD, USA) and LPMCs were collected after centrifugation at 300g for 10 min.
For splenocyte isolation, mouse spleens were cut into 3- to 5-mm pieces, and smashed using a syringe plunger and then filtered through a 70-μm cell strainer. Red blood cells were removed using red blood cell lysing buffer (Sigma, USA). Prepared single-cell suspensions were further processed flow cytometry analysis.

SCs were isolated as we described previously from 3-week-old male Kunming mice [32 (link),33 (link)]. In brief, the testes were aseptically isolated from mice, and washed 3 times in phosphate-buffered saline (PBS). Subsequently, the decapsulated testes were digested in Dulbecco’s Modified Eagle Medium (DMEM) (C11995500BT, Gibco, Grand Island, NY, USA) containing 500 μg/mL collagenase IV (C-5138, BioSharp, Beijing, China) at 37 °C by shaking in a water bath for 5–7 min. DMEM medium was used to terminate the digestion. After unit gravity sedimentation for 2 min, the Leydig cells enriched with supernatant were discarded. Then, the remaining tissues were digested in 0.25% trypsin (25200072, Gibco, Grand Island, NY, USA) at 37 °C for 10 min. The DMEM with 10% fetal bovine serum (FBS) (FSD500, ExCell Bio, Shanghai, China) was added to terminate the digestion. For separation and culture of SCs, the cell suspension was filtered through a 100-mesh filter screen, then the resultant filtrate was centrifuged at 1200× g rpm for 10 min to separate cells. SCs were cultured in DMEM with 10% FBS at 37 °C with 5% CO₂; 24 h later, after the cells had been PBS-washed twice, the fresh medium was added to continue the cell culture.