Gs 6r beckman centrifuge

The mouse model provides invaluable opportunities to explore consequences of altering the coding of specific genes on precise tissue functions. However, the small size of mouse testis allowed obtaining interstitial tissue- and seminiferous tubule-enriched fractions in small amounts. Mice were first anaesthetized (urethane, 1 g/kg IP, Sigma, St-Louis, MO, USA) before decapitation next, testes and epididymides were harvested. Blood was collected, allowed to clot; serum was obtained by centrifugation at 1500 rpm (GS-6R Beckman Centrifuge, JH-3.8 Rotor) 20 min and stored at -80C. Animal use protocol was approved by University of Montreal Animal Care Committee (Protocol number 12–126).

The interstitium and the seminiferous tubules of the testis have different topography and functions. Therefore, all our assays were performed on interstitium- and seminiferous tubule-enriched fractions. In addition, to maximize detection of phosphorylated protein forms within the samples under study, tissue fraction were not pre-exposed to enzymes and processed as described earlier (Akpovi and Pelletier, 2009 (link); Pelletier et al., 2015 (link)). Briefly, seminiferous tubules were mechanically teased apart from the interstitium from freshly decapsulated testes in cold phosphate buffered saline (PBS: 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4) containing 2 mM PMSF, 1 mM EGTA, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 4 mM Na₃VO₄, 80 mM NaF and 20 mM Na₄P₂O₇ with 10 μM bpV (phen). The resulting seminiferous tubule-interstitium suspension was centrifuged 15 min at 400 rpm (GS-6R Beckman Centrifuge, JH-3.8 Rotor) at 4°C after having been allowed to decant. The interstitial tissue- (ITf) (supernatant) and seminiferous tubule-enriched (STf) (pellet) fractions were centrifuged 10 min at 1,000 rpm (GS-6R Beckman Centrifuge, JH-3.8 Rotor) at 4°C. Each fraction was characterized under the light microscope as described earlier (Pelletier et al., 2018 (link); Akpovi and Pelletier, 2009 (link)).