br Cell viability proliferation assays br Cell viability and
2.3. Cell viability/proliferation assays
Cell viability and total cell number were assessed using 3-(4, 5-di-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Li et al.,
2007). Experiments were performed in 24-well plates. Cells were treated in 0.5 mL phenol red-free medium supplemented with 10% CS-FBS, respective for diﬀerent cell lines, as described in “Cell culture conditions”. Cells were treated for 1, 3, or 7 days as required by re-spective experiments. Cells were treated for 1 day, 3 days or 7 days. Medium was removed from the test wells at the end of treatment, and 0.5 mL freshly prepared treatment medium that contained 0.1% (w/v) MTT was added to each well. Cells were cultured at 37 °C for 2.0 h, 0.5 mL solublization solution (20% SDS in 0.02 M HCl in H2O) was added to each well and the plate swirled to mix thoroughly. The mixed content in the plates was incubated at 37 °C overnight, and the contents mixed again by vortexing. A 100 μl sample from each well was trans-ferred to wells of a 96-well plate, and absorbance read at 570 nm. Each experiment was repeated 4 times independently.
2.4. Androgen responsive element (ARE) – mediated luciferase reporter assay for AR activity
ARE-luciferase assays were performed to evaluate the AR stimula-tory eﬀect of androgens as described (Wu et al., 2011). Cells were seeded in 6-well plates with 3 × 105 LAPC-4 or VCaP Necrosulfonamide per well, and were incubated for 3 days and 7 days, respectively. Cells were trans-fected with the ARE-luciferase promoter-driven reporter plasmid using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM medium (Thermo Fisher Scientific) overnight following the manufacturer's in-struction. Transfected cells were trypsinized using trypsin-EDTA, wa-shed once with medium and plated in 24-well plates at 1.5 × 105 cells per well in culture medium supplemented with 10% CS-FBS. Cells were cultured for 1 day, and treated for 1 day to evaluate AR activity. After incubation cells were rinsed 3 times with phosphate-buﬀered saline PBS, and cells were lysed by addition of 100 μl 1X reporter lysis buﬀer (RLB) (Promega, Madison, WI) to each well. The plates containing ly-sates were shaken on a plate shaker at 250 rpm for 10 min, and frozen at −80 °C. The frozen lysates were thawed at room temperature, trans-ferred to clean Eppendorf tubes and centrifuged at 10,000x g for 10 min. A portion of the supernatant to be used for the luciferase assay was taken for protein determination using the BCA protein assay (Thermo Fisher Scientific) to obtain the total amount of protein (mg) in the lysate. Luciferase activity of the supernatant was measured using the Luciferase Assay System reagent (Promega). Luminescence was measured using a luminescence plate reader. The luciferase activity was expressed as relative luminescent unit (RLU) per μg of protein. Ex-periments were set up in 4 replicates, and luciferase activity in each sample was analyzed in triplicate.
2.5. Ex vivo experiments using fresh clinical human prostate tissue specimens
Metabolism of DHEAS and DHEA to DHT in intact tissue specimens was evaluated ex vivo in fresh remnant prostate tissue specimens. Fresh prostate tissue specimens were received in phenol red-free RPMI1640 supplemented with 10% CS-FBS. Experiments were set up within 3 h of reception of the tissue. Experiments were set up in 300 μl medium per well in 48-well plates. Tissue specimens were cut into 2–3 mm3 pieces and 2–3 pieces of tissue were transferred into each well in 48-well plates. Upon the transfer of tissue specimens into the wells, all liquid ovulation accompanied the tissue specimens was removed using sterile pip-ette tips and blotting with sterile Q-tips, and the tissue was rinsed 3 times with phenol red-free RPMI1640 supplemented with 10% CS-FBS. Residual medium was fully removed as described previously before 300 μl of phenol red-free RPMI1640 supplemented with 10% CS-FBS containing respective treatment agents was added into the well. Tissue specimens were incubated for 1 or 4 days during which medium sam-ples were harvested and stored at −80 °C. At the end of treatment, the tissue specimens in each well were transferred to an Eppendorf tube which was weighed individually, and the residual liquid was removed Molecular and Cellular Endocrinology 486 (2019) 79–88
thoroughly using pipette tips and blotting with Q-tips before the tissue and the tube were weighed together. Final tissue weight was reached by subtraction of the weight of the empty Eppendorf tube from the total weight of the tube and the tissue. DHT or DHEA in culture medium was measured using ELISA, and the weight of tissue in each sample (g) was used to normalize DHT or DHEA production.