Brdu cell proliferation elisa

Cell proliferation was measured using either a Cell Proliferation BrdU ELISA (Roche Diagnostics Ltd., Sussex, UK) according to the manufacturer's instructions, or by a Resazurin reduction based assay (66 ).
For BrDU based assays, cells (JU77, Met5A, and NCI-H226) were seeded at 2.5 × 10³/well in a 96-well plate. Following overnight incubation, cells were treated for 24 or 48 h with human recombinant MST1 (0–250 ng/mL), LCRF-0004 (0–200 nM), RON8 (200–1,000 ng/mL), or NRWHE (25–200 ng/mL) or combinations of drug as appropriate. Absorbance was measured on a plate reader at 450 nm with a reference wavelength set to 690 nm. Untreated wells were used for normalization purposes and set to 100%.
For Resazurin based assays cells (LP9, NCI-H226, and REN) were seeded at 3.5 × 10³/well in a 96 well plate. Following overnight incubation, cells were serum depleted by replacing the media with fresh media containing 0.5% FBS and left for a further 24 h. Subsequently, cells were treated for 72 h with combinations of drug as appropriate. Resazurin was added after the 72 h treatment period, and following incubation for approximately 4 h at 37°C, fluorescence was measured on a fluorescence plate reader using a 560 nm excitation/590 nm emission filter set.

Cell proliferation was measured using the Cell Proliferation BrdU ELISA (Roche Diagnostics Ltd., UK), according to manufacturer's instructions. Briefly, cells (H460, H1299 and SKMES-1) were seeded at 2.5 × 10³/well in a 96-well plate. Following overnight incubation, cells were treated for 72hr with cisplatin (0-100μM) alone, or in combination with DEAB (15μM) or equimolar concentrations of Disulfiram (0.25μM) and copper chloride (0.25μM) (DSF/CuCl₂). Absorbance was recorded at 450 nm and sensitivity to cisplatin was calculated based on the percentage cell proliferation relative to untreated controls, which were set at 100%.

Cell viability was determined after a 24-h, 48-h or 72-h incubation of INS1E cells with cytokines using a microplate-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 562/650 nm^{33 (link)}. Caspase-3 or −12 activation was quantified using a green caspase-3 or red caspase-12 staining kit (PromoCell, Heidelberg, Germany) according to the instruction manual, followed by data analysis by FlowJo software (Tree Star, Ashland, OR)^{3 (link),34 (link)}. In the case of human EndoC-βH1 beta-cells the MTT assay does not provide reproducible results and therefore the PI method was used as in the case of other studies using this human beta-cell line^{35 (link)–37 (link)}. The percentage of dead human EndoC-βH1 beta-cells (at least 500 cells per each condition) was determined after a 15-min incubation with the DNA-binding dye propidium iodide (PI) (50 µg/ml). The proliferation rate was quantified by using the Cell Proliferation BrdU–ELISA (Roche, Mannheim, Germany)^{3 (link)}.