Transfer all medium from each well to 1.5 ml tubes. Centrifuge at high speed 10000g for 1 min in Eppendorf tube. Transfer supernatant to 1.5 ml tube and snap frozen in liquid nitrogen then kept at -80 °C until hormonal analysis.
Trypsinization were used for harvesting live cell, 500 µl of pre-warmed trypsin-EDTA were added to each well and incubated at 37.5 °C for 5 min. Then dislodged cells from well by pipetting and to stop trypsin effect, 100 µl of heated inactivated fatal calve serum (HI FCS) were added to each well and mixed well. Transfer cell suspension into 1.5 ml Eppendorf tube, after that rinse the well by 500 µl of pre warm dPBS and combined with previous cell suspension. Centrifuge cell suspension at high speed for 2 minute. Remove the supernatant and re-suspended pellet in 250 µl pre-warmed 1x dPBS. Manually agitating the tube by hand “finger flick” and mixed well by pipetting up/down gently. An aliquot of 10 µl cell suspension was taken for cell viability. The remaining cells were centrifuged for 2 min at 10000g, Remove supernatant and freeze pellet in liquid nitrogen then tacked out from liquid nitrogen and kept at -80°C until RNA extraction. The number of the GCs was determined by adding 10 µl of trypan blue to 10 µl of cell suspension and counted the cell number under microscope …show more content…
The assay was performed as described in section 3.2.2.2. P4 concentrations were analysed in spent culture media for cells cultured for 48, 96 and 144 hr in the present of melatonin 0, 20, 200 and 2000 pg/ml under both 5% O2 and air condition. Samples were diluted 100 fold in PBS and analysed in duplicate with standard (0, 0.5, 1, 2, 5, 10 and 20ng/ml), blank and quality control. To avoid variation among plates, all samples were run together. Inter and intra assay % CV was (11.1) and (5.1) respectively.
4.2.3.2 Oestradiol
Trypsinization were used for harvesting live cell, 500 µl of pre-warmed trypsin-EDTA were added to each well and incubated at 37.5 °C for 5 min. Then dislodged cells from well by pipetting and to stop trypsin effect, 100 µl of heated inactivated fatal calve serum (HI FCS) were added to each well and mixed well. Transfer cell suspension into 1.5 ml Eppendorf tube, after that rinse the well by 500 µl of pre warm dPBS and combined with previous cell suspension. Centrifuge cell suspension at high speed for 2 minute. Remove the supernatant and re-suspended pellet in 250 µl pre-warmed 1x dPBS. Manually agitating the tube by hand “finger flick” and mixed well by pipetting up/down gently. An aliquot of 10 µl cell suspension was taken for cell viability. The remaining cells were centrifuged for 2 min at 10000g, Remove supernatant and freeze pellet in liquid nitrogen then tacked out from liquid nitrogen and kept at -80°C until RNA extraction. The number of the GCs was determined by adding 10 µl of trypan blue to 10 µl of cell suspension and counted the cell number under microscope …show more content…
The assay was performed as described in section 3.2.2.2. P4 concentrations were analysed in spent culture media for cells cultured for 48, 96 and 144 hr in the present of melatonin 0, 20, 200 and 2000 pg/ml under both 5% O2 and air condition. Samples were diluted 100 fold in PBS and analysed in duplicate with standard (0, 0.5, 1, 2, 5, 10 and 20ng/ml), blank and quality control. To avoid variation among plates, all samples were run together. Inter and intra assay % CV was (11.1) and (5.1) respectively.
4.2.3.2 Oestradiol