Isolation of Immune Regulatory Sertoli Cells from Mouse Seminiferous Tubules

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…

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.