Cell proliferation elisa brdu

PBL from patients and controls were isolated via centrifugation on a Ficoll-Hypaque gradient for 20 min, washed twice, and resuspended in Roswell Park Memorial Institute 1640 medium with HEPES, supplemented with 2 mM glutamine (Sigma-Aldrich Japan, Tokyo, Japan), 100 units/ml penicillin G (Sigma-Aldrich Japan, Tokyo, Japan), 100 μg/ml streptomycin sulfate, and 10% heat-inactivated human AB serum (Biowest, Nuaillé, France). Cells (1 × 10⁵/well) were incubated in a total volume of 100 ml with the peptides or concanavalin A (4 μg/ml, Fujifilm Wako Pure Chemical Co., Osaka, Japan) in triplicates in 96-well round-bottom culture plates (Nunc, Roskilde, Denmark) at 37°C in a humidified atmosphere with 5% CO₂. The cultures were incubated for 4 days and then pulsed with bromodeoxyuridine (BrdU) during the last 16 h of incubation. Cellular proliferation was assessed using a BrdU enzyme-linked immunosorbent assay kit (Cell Proliferation ELISA BrdU, Sigma), according to the manufacturer’s instructions. After co-culture with Brdu for 16 h, the BrdU labeling solution was removed and anti-BrdU antibody was added. The OD at 450 nm for BrdU was measured using a luminescence plate reader (ARVO X3, Perkin Elmer Japan, Yokohama, Japan). The results were expressed as stimulation index (SI: mean OD_450nm in stimulated cultures / mean OD_{450 nm} in unstimulated control cultures).

The human prostate carcinoma DU145 (not detectably hormone sensitive, ATCC®HTB-81™) and LNCaP cell lines (androgen receptor, positive; estrogen receptor, positive; ATCC®CRL-1740™) were purchased from the American Type Culture Collections. The human normal prostate PNT-2 cell line was purchased from HPA Culture Collections (Sigma-Aldrich, MO, USA). Cells were cultured in appropriate RPMI 1640, medium (Sigma-Aldrich, MO, USA) according to the ATCC protocol with an addition of 10% FBS (Sigma-Aldrich, MO, USA). Cells were seeded on 96-well plates (8 × 10⁴ cells per well). 24 h after seeding, growth medium was replaced with a medium containing juices of young shoots or the vegetable at full maturity. Percentage dilution of juices were prepared in the appropriate cell culture medium and added to the wells on growth plates. The final applied concentrations of each treatment were 1, 2, 3, 4 and 5% in cell culture medium for 24, 48 and 72 h of incubation. Untreated cells, growing in culture medium, without any of concentration of juices, were used as a negative, untreated control. Cell proliferation was determined with Cell Proliferation ELISA, BrdU (Sigma-Aldrich, MO, USA), according to producing instruction.

The proliferation of the organoids NL, A, AT, Ade^CIN TS and Ade^CIN TSK after 7 days of culture was measured using the Cell Proliferation Elisa BrDU (colorimetric) kit purchased from Sigma-Aldrich (Saint Louis, MO, USA) as described [13 (link)]. Values were normalized to total cellular protein concentration. The proliferation rate of Ade^CIN TS and Ade^CIN TSK incubated with various concentrations of HMPSNE or AOAA was determined. Proliferation assays were performed at least 4 times in duplicates.