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Preparation of Primary Leydig Cell Culture From Mouse Testis

First, testes from 3 week old mice were harvested in sterile conditions. The testes were then decapsulated in a laminar flow hood. The seminiferous tubules were teased apart, releasing Leydig cells and germ cells. The tubules were then discarded leaving free Leydig cells and some germ cells in the solution. Next, the cells were centrifuged and washed. The remaining Leydig cells were plated in fibronectin treated 48 well plates and incubated at 32°C with 5% CO2 for 48 hours. The medium was then changed every 24 hours to remove unattached germ cells. The cells were lysed and the protein was extracted for use in a western blot analysis.