Cultures of Sertoli cells isolated from 20-day-old mice are widely used in research as substitutes for adult Sertoli cell cultures. levels of triacylglycerols, cholesteryl esters, and seminolipid, and the proteins in their spent medium were mainly engaged in cellular metabolism. In contrast, proteins involved in cell division, including anti-Mullerian hormone, cell division cycle protein 42 (CDC42), and collagen isoforms, were at higher levels in Sertoli cell cultures derived from 20-day-old mice. Therefore, cultured Sertoli cells derived from 10-week-old mice, rather than those from 20-day-old animals, should be used for studies on properties of adult Sertoli cells. and knockout mice (37), it is important that Sertoli cells can be isolated with high purity from adult animals for further studies. To date, only a limited number of reports describe protocols to isolate and culture Sertoli cells from adult rats/mice, and complete information on yield and purity has not yet been available (38C44). Thus, the second objective of our report is to describe this protocol with yield and purity results on Sertoli cell isolation from 10-week-old mice. Materials and Methods Animals CD-1 male mice, 20 days old and 10 weeks old, obtained from Charles River laboratories (Senneville, QC, Canada) and kept in a JNJ-64619178 temperature-controlled (22.5C) room with a 12-hour light and 12-hour dark photoperiod, were fed with Purina rodent chow and water. The use and handling of mice for all those experiments adhered to the Canadian Council on Animal Care guidelines, with approval by the University of Ottawa Animal Care committee, which endorses the use of ARRIVE checklists and guidelines. Mice were sacrificed by cervical dislocation for testis collection. Sertoli cell JNJ-64619178 culture As described previously (41, 46, 47), Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (DMEM-F12) supplemented with gentamycin (20 g/mL), bacitracin (10 g/mL) and fungizone (0.25 g/mL) was used as isolation medium to prepare loose cells (Sertoli cells, testicular germ cells, and other co-isolated cells) from seminiferous tubules (prepared by denuding collected testes free of the tunica albuginea), whereas COL12A1 culture medium, which was isolation medium supplemented with insulin (10 g/mL), transferrin (5.5 g/mL), sodium selenite (0.0067 g/mL), and epidermal growth factor (2.5 ng/mL), was used for culturing Sertoli cells. Loose cells were generated from seminiferous tubules through JNJ-64619178 serial digestions with various enzymes (made in isolation medium), as previously described (39, 41, 48, 49), although we empirically decided the sequence of the sets of enzymes used, as described below, to give the highest yield of Sertoli cells. Isolation medium was used to prepare the 3 mixtures of the enzyme solutions (see below). For the last mixture (collagenase (1 mg/mL), hyaluronidase (2 mg/mL), DNase I JNJ-64619178 (50 g/mL), and soybean trypsin inhibitor (SBTI) (0.1 mg/mL), the pH of the solution was slightly adjusted to be 7.4 with 1 N NaOH. All actions in Sertoli cell isolation and culture were performed at 35C. For the protocol described below, 5 pairs of testes from 20-day-old mice and 3 to 5 5 pairs of testes from 10-week-old mice were used. Tissues/cells were processed in 50-mL Erlenmeyer flasks freshly coated with Millipore-Sigma Sigmacote solution, following manufacturers instruction. Sources of medium, reagents, enzymes, JNJ-64619178 and specific plasticware used in Sertoli cell isolation are described in Reference (45). All enzymatic treatments were performed in a shaking water bath, with continuous movement of 150 revolutions/minute. This was begun by treating (15 minutes) the testes denuded of tunica albuginea with the mixture of collagenase Type I (0.5 mg/mL) and DNase I (50 g/mL) to disperse the seminiferous tubules, thus releasing interstitial cells into the surrounding. The supernatant was removed from the seminiferous tubules, which sedimented by gravity. The.