Sertoli cell isolation was carried out as described [32 (link), 33 (link)] with some modifications. Briefly, testes of adult mice were decapsulated in HBSS containing collagenase (1 mg/ml) for 20 min and seminiferous tubules were collected by sedimentation. Then, seminiferous tubules were dispersed in a HBSS solution containing collagenase (1 mg/ml)/hyaluronidase (1 mg/ml)/DNase (0.4 mg/ml) for 20 min at 34 °C. After washing with PBS, an additional digestion step was performed with accutase cell dissociation reagent (Innovative Cell Technologies, USA) for 15 min at 34 °C. The tubular pellet was washed with PBS and Sertoli cells were freed from the seminiferous epithelium by resuspending the pellet in DMEM/F12 medium containing 5% FBS. Myoid cells were removed by differential adhesion at the first 30 min and Sertoli cells were further cultured overnight at 34 °C. The main germ cell fraction in the supernatant was pelleted by centrifugation at 300 g. During Sertoli cell culture, contaminating germ cells were removed by hypotonic shock.
Accutase cell dissociation reagent
Manufactured by Innovative Cell Technologies
Sourced in United States
Accutase is a cell dissociation reagent. It is a mixture of proteolytic and collagenolytic enzymes formulated for gentle and effective dissociation of adherent cells.
Automatically generated - may contain errors
3 protocols using accutase cell dissociation reagent
1
Isolation of Murine Sertoli Cells
2
Primary Sertoli Cell Culture from Mice
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Primary Sertoli cell culture was carried out as described previously [49, 50] with some modifications. Briefly, to obtain Sertoli cells for primary culture, testes of 3-week-old mice were decapsulated in HBSS and seminiferous tubules were dispersed in a HBSS solution containing collagenase (0.1%)/hyaluronidase (0.1%)/DNase (0.04%) for 20 min at 34 °C. After washing with PBS, an additional digestion step was performed with Accutase cell dissociation reagent (Innovative Cell Technologies) for 15 min at 34 °C. The tubular pellet was washed with PBS, and Sertoli cells were freed from the seminiferous epithelium by resuspending the pellet in Dulbecco's modified Eagle's medium (DMEM)/ F12 medium. Cells in the supernatant were collected and cultured in DMEM/F12 medium containing 5% FBS overnight at 34 °C. Following attachment of the Sertoli cells to the bottom of the tissue culture plate, they acquired an irregular shape, whereas the germ cells remained suspended and were easily removed by repeated washing. The purity of primary Sertoli cells was routinely analyzed by immunofluorescent staining with SOX9.
3
Isolation and Culture of IMC-1 Cell Line
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The biopsied tissue was minced using a scalpel and agitated in Eagle’s minimum essential medium (E-MEM; Wako Pure Chemical Industries, Osaka, Japan) containing 1 mg/mL collagenase type I (Sigma-Aldrich Japan, Tokyo, Japan) at 37.5°C for 1 h. After the mixture was filtered using a 100 μm nylon mesh, it was cultured in Roswell Park Memorial Institute medium (RPMI-1640, Wako Pure Chemical Industries) supplemented with 3% foetal bovine serum (FBS), amino acid supplement (GlutaMAX, Thermo Fisher Scientific, Waltham, MA, USA), and antibiotic-antifungal solution (Penicillin-Streptomycin Mixed Solution; 100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B; Nacalai Tesque, Kyoto, Japan) at 37.0°C under 5% CO2. After 24 h, cells adhering to the dish were observed. Subsequently, the medium was changed once every two days until the cells were 90% to 100% confluent. Accutase cell dissociation reagent (Innovative Cell Technologies, San Diego, CA, USA) was used for subculturing. The cells were passaged 100 times and stable cell proliferation was confirmed. This cell line was named IMC-1.
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