decapsulated and digested enzymatically with trypsin and deoxyribonuclease for 30 min at 37 C and centrifuged at 750 g for 5 min The pellet was mixed soybean trypsin inhibitor Raf Inhibitors then centrifuged and incubated with collagenase and hyaluronidase for 30 min at 37 C After incubation this fraction was centrifuged (10 min at 40.g) The pellet was taken to isolate Sertoli cells and supernatant was discarded After counting Sertoli cells were plated in multiwell plates (3.105 cells/cm2) in Medium 199 pH 74 1% FBS and maintained in humidified 5% CO2 atmosphere at 37 C for 24 h for attachment The medium was then changed to serum-free medium and cells were maintained for more 24 h Medium was then replaced by fresh medium containing treatments and cells were incubated for more 24 h Morphology was examined at the end of the 24 h treatments Medium was replaced by fresh serum-free medium and treatments were immediately initiated by adding concentrated solutions of retinol (dissolved in ethanol) or Trolox (dissolved in water) to reach final concentrations in the well .

The final ethanol concentration did not exceed 02% in any experiment Vehicle controls with this concentration of ethanol were performed for each condition showing no alterations At the end of 24 h of treatments under the conditions mentioned above cells were used for Bosutinib assay by the following procedures: for DCFH-DA assay incubation medium was replaced by the fresh medium containing 1% FBS and DCFH-DA 100 M and assayed as described below For immunoblot retinol incubation was stopped by removal of the incubation medium and addition of Laemmli-sample buffer followed by the procedures described below at °immunoblot subsection For viability measurements at the end of 24 h of retinol treatment MTT was added to the wells and the MTT assay was performed as described below Sertoli cells cultures were estimated to be 90C 95% pure as assessed by the alkaline phosphatase assay 23 DCFH-DA assay .

Intracellular reactive species production was determined by the DCFH-DAbased real-time assay using intact living cells (Wang and Joseph 1999) Briefly Sertoli cells were plated onto 96-well plates incubated with retinol for 24 h After that the medium was changed for 1% FBS culture medium with DCFH-DA 100 M (stock solution in DMSO 10 mM) and cells were incubated at 5% CO2 and 37 C for DCFH-DA loading Then cells were washed PBS was added to each culture well and the cells were placed in the microplate fluorescence reader (F2000 Hitachi Ltd Tokyo Japan) Changes in the fluorescence by the oxidation of DCFH into the fluorogen DCF were monitored during 1 h at 37 C A positive control for Hematomas intracellular reactive species production was performed with H2O2 1 mM Excitation filter was set at 485 À 10 nm and the emission filter was set at 530 À 125 nm Data were recorded every 30 s and plotted in Excel software 24 Immunoblot To perform immunoblot experiments Sertoli cells were lysed in Laemmlisample buffer (625 mM TrisCHCl pH 68 .