leydig cell from testes
I) Removal of testes: Put into ice-chilled D-PBS.
II) Cannulation: Perfuse 0.5 ml DB (1 mg per ml collagenase + one 1 ml aliquot of 2.4 U/ml dispase, final conc. = 0.185 U/ml in 13 ml) into testicular artery of each testis. Preparation of internal collagenase: 12 mg of collagenase + one 1 ml aliquot dispase + 12 ml of DB.
III) Put each perfused testis into beaker containing fresh, chilled Dulbecco’s PBS (D-PBS).
IV) Decapsulation: Remove the tunica of each testis. Place 2 testes into a 50 ml centrifuge tube containing 5 ml DB. Repeat for remaining 5 pairs of testes.
V) Dissocation: Add 5 ml external collagenase in to each testis containing tube (results in 0.25 mg/ml collagenase). Shake @ 70 to 80 cycles per minute @ 34_ C for 10 to 20 minutes (monitor). Preparation of external collagenase: 16 mg of collagenase + 32 ml of DB + one 0.42 mg aliquot of DNase (Roche, # 104 159) + one 1ml aliquot dispase.
VI) Centifugation: Bring volume of each tube up to 50 ml with SB. Recap tube and invert the tube several times. Let the Seminferious tubules settle for 1 to 2 minutes. With a 10 or 25 ml pipette, draw up the supernate, without disturbing the pellet. Save the supernate. Once the supernate has been collected, refill the tube containing the tubules with SD again invert the tube, then allow the tubules to settle, and collect the supernate. Repeat 3 to 4 more times. Spin down the tubes (Falcon 225ml tubes; Falcon# 352075) at 800xg for 20 minutes. Keep the pellets.
VII) Percoll density gradient centrifugation: 8 ml 10X HBSS + 88 ml Percoll (= stock isotonic Percoll, SIP). 96 ml SIP + 80 ml PB = 175 ml of 55% pH 7.4 SIP [this is enough SIP for one tube]. To this tube (Nalgene 175ml conical PC centrifugation bottle # 3144-0175) add five aliquot of DNase along will the cell pellets. To the dummy tube (175 ml of 55% pH 7.4 SIP) pipet 25 ul’s each of density marker beads (DMB) 3, 4, & 5 [since Percoll is expensive you can save the dummy tube or forgo it]. Balance the tubes and, using a Beckman JA-10 rotor (use adapters and cushions), spin @ 10,000 RPM (approx. 17,690 x g) for 40 minutes at 4_ C. Pipet off the upper layer of the gradient carefully, from the meniscus to DMB 4, discard this layer. Pipet off the upper layer from DMB 4 to the midpoint between DMB’s 4 & 5, discard this layer. Collect the fraction from the midpoint to the bottom of the gradient with a pipet carefully avoiding the red blood cell clumps. Fill both tubes (225ml Falcon) with PB to dilute out the Percoll and centrifuge @ 800xg for 20 minutes. Keep the cell pellet.
VIII) BSA Multi-Gradient centrifugation: Carefully and slowly layer 50 ml of 5% BSA –PB (made by mixing 25 ml 10% BSA-PB + 25 ml 0% BSA-PB) on top of 65 ml of 10% BSA-PB. Then layer 50 ml of 2.5% BSA–PB (made by mixing 12.5 ml 10% BSA-PB + 37.5 ml 0% BSA-PB) on top of the 5% BSA-PB layer. Finally layer 10 ml of 1% BSA-PB/Cell mixture on top of the 2.5% BSA-PB layer (1% BSA-PB/Cell mixture made by mixing the cell pellets with 1 ml 10% BSA-PB + 9 ml 0% BSA-PB). Spin down the tubes (Falcon 225ml tubes; Falcon# 352075) at 50xg (550 rpm) for 10 minutes. Collect the bottom 50 ml, fill up the 225 ml tube with 0% BSA-PB to dilute out the high % of BSA and centrifuge @ 200xg for 15 minutes. Keep the cell pellet.
IX) Final pellet: Resuspend the final pellet in 2-4 ml of DB or LCM. Perform a hemocytometer count (the total number of cells falling within the boundaries of 5 squares on a Neubauer hemocytometer X .05 = the number of cells in millions per ml) to obtain yield.