Isolation of Immune Regulatory Sertoli Cells from Mouse Seminiferous Tubules
Published: August 31, 2023
Abstract
Source: Bhushan, S. et al., Isolation of Sertoli Cells and Peritubular Cells from Rat Testes. J. Vis. Exp. (2016).
This video demonstrates the isolation procedure of Sertoli cells from mouse seminiferous tubules, a component of the testes. Enzymatic digestion and repeated washes promote the release of Sertoli cells into the solution, which can be cultured further for proliferation and growth.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of Media, Enzyme Solutions, and Animals Prepare 1% iodine alcohol by dissolving 1 g of iodine in 100 ml ethanol. Prepare 2x 500 ml Dulbecco´s phosphate buffered saline (PBS) without Ca2+ and Mg2+ supplemented each with 7.5 ml D-glucose (100 g/L) and 5 ml 100x Pe…
Representative Results
Figure 1. Testes from Wistar rats are excised and decapsulated. (A) The peritoneal cavity is opened through a longitudinal incision as described in the text. An asterisk (*) indicates the epidydimal fat pad. (B) Testes are removed by cutting the spermatic cord and (C) collected in a 50 ml conical tube containing 20 ml PBS. When all testes have been collected they are d…
開示
The authors have nothing to disclose.
Materials
| Corning™ Cell Strainers 70 µm | Corning | 431751 | White color |
| DAPI mountant ProLong® Gold Antifade | Life Technologies | P-36931 | |
| D-glucose | Sigma | G8644 | 100 g/l |
| Dulbecco's PBS without Ca2+/Mg2+ | Gibco | 14190-094 | |
| DNase I | Roche | 10104159001 | |
| Hyaluronidase from bovine testis | Sigma | H3506 | |
| Penicillin (5,000 U/ml) Streptomycin (5,000 µg/ml) | Gibco | 15070-063 | 100x solution |
| RPMI-1640 | Gibco | 21875-034 | Contains 300 mg/L L-glutamine |
| Trypsin from porcine pancreas | Sigma | T5266 | |
| Trypan Blue Stain (0.4%) | Gibco | 15250-061 | |
| Trypsin inhibitor from soybean | Sigma | T6522 | The Sigma product is considerably cheaper than the previously used BPTI (Aprotinin) from Roche. |
| Wistar WU rats | Charles River | N/A | Should be 19 days old on day of experiment. |
