Sonic dismembranator model 300

Cells from the anterior pituitary FS cell line TtT/GF were initially provided by Dr. U. Renner (Max-Planck-Institute of Psychiatry, Dept. of Endocrinology, Munich, Germany). TtT/GF cells exhibit the morphological, biochemical and physiological features of typical FS cells [7 (link);11 (link)]. The cells were grown in DMEM supplemented with 5% fetal calf serum, 3.7 g/ml NaHCO₃, 10 mM HEPES, pH 7.2, and antibiotics at 37°C under a 95%-5% air-CO₂ atmosphere. For immunofluorescence studies, the cells were seeded on glass coverslips (No. 0 thickness). Cells were serum-starved for 24 h prior treatment with bFGF (15 ng/ml).
Cells were harvested once they had reached 60–70% confluence. After a wash with cold phosphate buffered saline (PBS: 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄ and 1.5 mM KH₂PO₄, pH 7.4), the cells were detached and recovered by centrifuged at 2,000 RPM for 5 min in a Beckman, GS-6R centrifuge with a GH 3.8 rotor (Beckman Coulter Canada Inc., Mississauga, ON, Canada). The cell pellet was resuspended in a protease phosphatase inhibitor cocktail made in PBS (4mM Na₃VO₄, 80 mM NaF, 20 mM Na₄P₂O₇, 10 μM bpV-phen, 5 μg/ml leupeptin, 5 μg/ml aprotinin and 2mM EGTA, pH 8.5), except for the alkaline phosphatase studies, and sonicated at moderate intensity when needed (Fisher Sonic Dismembranator Model 300) for 30 seconds.

Sertoli cells cultured to ~70–80% confluence on culture day 7 in a 6-cm plate were washed twice with PBS containing 100 μM of diethylenetriaminepentaacetic acid (DTPA, Sigma) (PBS/DTPA, 3 mL each wash). The Sertoli cell culture was then incubated (30 min, 35 °C, 5% CO₂) with 3 mL of Gibco HBSS (Thermo Fisher Scientific, A14026-076) containing 100 μM of DTPA (HBSS/DTPA) and 30 μM of DHE (Sigma). At the end of the incubation, the Sertoli cell culture was washed twice with PBS/DTPA (3 mL each wash) and the cells were detached from the plate by treatment (5 min, 35 °C, 5% CO₂) with 3 mL of Accutase. Sertoli cells were collected in a 15-mL tube and washed twice in PBS-DTPA (3 mL each wash) by centrifugation (800× g, 3 min, 28 °C). The Sertoli cell pellet was added with 1 mL of acetonitrile and the mixture was sonicated in a cup using a Fisher Sonic Dismembranator Model 300 at 90% output for 3 min. Following centrifugation at 12,000× g for 10 min at 4 °C to pellet cell particulates, the cell extract in the supernatant was transferred to a fresh tube and dried under vacuum. The dried cell extract was kept at −80 °C in the dark until HPLC analyses.