We try to performed some experiments in vitro, including transfection, in testicular Leydig cells
Hi Sir, We have isolated Leydig cells from 21, 35 and 60 day old rats which are called as progenitor, immature, and mature leydig cells respectively. Look for references anbalagan et al or Sriraman et.al but we have not isolated fetal Leydig cells. Any thing else pl. write to [email protected]
Hi there!
I have protocols for Leydig cell precursors isolation at rat day 13 pp, call me at 912071458 or email me at [email protected] if you want them.
Hello!!
Please find requested protocol which my colleague uses in his lab and forwarded me.
Protocol Isolation of Leydig cells:
Testes were decapsulated from a 60-day old rat and incubated in 50-ml tube containing 10-ml MEM-BSA 0.1% with 0.25 mg/ml Collagenase. Tube was incubated longitudinally at 100cycle/min, for 20 min at 37°C. The suspension obtained was filtered through strainer (70 Jim) ( BD ) and crude interstitial cells were collected by centrifugation at 300 g for 10 min and then washed twice in 0.1% (w/v) BSA in MEM. To obtain purified Leydig cells, 4 m1 of the crude cell suspension was loaded on top a discontinuous Percoll gradient (20, 40, 60 and 90% Percoll in HBSS) and centrifuged at 800 x g for 20 min. The fractions enriched in Leydig cells were then centrifuged with 60% Percoll at 14,000 x g for 30 min at 4°C. After centrifugation, purified Leydig cells were recovered from Percoll fractions heavier than 1.068 g/ml, discard the upper fraction and dilute the purified Leydig cells with MEM-BSA (up to 50 ml), centrifuge 350 g for 8 min at 20 C°. Wash the cells twice to remove Percoll. Purified Leydig cells were dissolved in 4 ml culture medium and counted by using phase-contrast objective (40X). Check the viability by using Trypan blue .
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