Cell proliferation elisa brdu assay

GDC-0941 and GDC-0973 were obtained from the chemistry department at Genentech, Inc. as a 10 mm DMSO stock solution. Cell lines were maintained at 37 °C and 5% CO₂ in RPMI 1640 medium with 10% fetal bovine serum, 2 mml-glutamine, and penicillin-streptomycin. Cells were plated in normal growth medium at 6000 cells/well in 96-well clear-bottom black plates. The following day, cells were transfected with 100 nm siRNA using DharmaFECT 3 (Dharmacon, Chicago, IL). 48 h post-transfection, cells were treated with GDC-0973 and GDC-0941. For cell proliferation assays, 10 nm GDC-0973 and 100 nm GDC-0941 were used. For cell death assays, 20 nm GDC-0973 and 200 nm GDC-0941 were used. 24 h following drug treatment, cell proliferation was measured using the Cell Proliferation ELISA BrdU assay (Roche Applied Science), and cell death was quantified using the Cell Death Detection ELISA PLUS assay (Roche Applied Science) according to manufacturer's instructions.

To evaluate the impact of VIRMA knockdown in PC-3, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay was performed. To determine differences in cell proliferation the Cell Proliferation ELISA BrdU assay (Roche Applied Sciences, Basel, Switzerland) was used. Invasion ability of cells was analyzed using BD Biocoat™ Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA). The procedures followed the published report [23 (link)]. For wound-healing assay cells were seeded and grown to 100% confluence and then scratched with a sterile 200 μL pipette tip to create an artificial wound. At 0, 4, 8, and 12 h after wounding, phase-contrast images of the wound healing process were photographed with a 10x objective lens. Eight images per condition were analyzed to determine averaging position of the migrating cells at the wound edges.

Cell viability and proliferation were assessed in 96-well culture plates with cells plated at a concentration of 4 × 10³ per well. After 24 h cells were incubated for 48 h with YM155, M4N, AT406 or vehicle control at equimolar concentrations. To assess cell viability and proliferation, CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) at an absorbance at 490 nm or Cell Proliferation ELISA, BrdU assay (Roche Applied Science, Mannheim, Germany) at an absorbance at 370 nm were performed according to the manufacturer’s protocol. All assays were analyzed using an Infinite® 200 microplate reader (Tecan Group Ltd., Crailsheim, Germany).
For cell cycle analysis, FTC cell lines were harvested, washed in ice cold PBS and resuspended in 80% ethanol. After incubation for 2 hours, cells were washed in PBS and RNAse A (100 µg/ml) together with propidium iodide (PI; 50 µg/ml) was added. Finally, cells were analyzed by Fluorescence-activated cell sorting (FACS) after 30 minutes of incubation at 37 °C using a BD FACSCanto™ II (BD Biosciences, San Jose, CA, USA).
Apoptotic cell death was quantified by using the FITC Annexin V/Dead Cell Apoptosis Kit (Molecular Probes, Eugene, OR, USA) and Caspase-Glo® 3/7 Assay (Promega Corp, Mannheim, Germany) according to the manufacturer’s protocol.

ASCs were trypsinized and the number of live cells was counted using Trypan Blue exclusion dye. Proliferation was assessed using the Cell Proliferation Elisa BrdU assay as described by the manufacturer (Roche SAS, Boulogne-Billancourt).

For proliferation assay, Cell Proliferation ELISA, BrdU assay (Roche Applied Science, Penzberg, Germany) was used following the manufacturer’s instruction. For knockdown experiments the data were normalized with the scrambled control. For cell viability assay, cell proliferation reagent WST-1 (Roche Applied Science) was used following the manufacturer’s instruction. All experiments were performed in triplicates. For clonogenicity assay, 2000 cells were seeded on 6-well plates in triplicates. Cells were cultured with complete medium and allowed to grow until clones were visible. Cells were stained with crystal violet and clones were quantified. For asymmetrical division analysis, cells were stained with PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions. At day 0, unstained and PKH26-stained cells were assessed with flow cytometry after which PKH26 staining was visualized at day 1, 3, 7, and 14. For cell cycle analysis, siRNA-treated cells were harvested after 24, 48, and 72 h and fixed in ice-cold 70% ethanol. Cells were resuspended in propidium iodide solution (PBS, 1% Triton X-100, 100 mg/mL RNaseA, 1 mg/mL PI), and processed for FACS analysis as previously described.