Bio tek elisa reader el 800

Viability/proliferation of 8932 pancreatic cancer cells and MCF-7 breast cancer cells was evaluated using a WST-1 proliferation assay (Roche Diagnostics). For quantification 2.5 x 10³ cells were seeded in triplicates in 96-well plates and were left to adhere overnight. Metabolic activity of cells, indicative of cell proliferation/viability, was assessed after addition of 10 μl (per 100 μl medium) of a liquid reagent containing tetrazolium salt to the respective cell medium. Changes in formazan dye generation were measured in an ELISA reader after 30 min of incubation at 37°C and 5% CO₂ (Bio-Tek ELISA Reader EL 800, Bio-Tek Instruments GmbH, Bad Friedrichshall, Germany). Results of absorbance measurements (450 nm) of both cell lines were plotted in one graph to display relative metabolic activity of both cell lines.

To determine the concentration of lactate in the supernatant of the MCF-7 and 8932 tumor cells, the cell medium was collected following adhesion of the cells overnight at 37 °C and 5 % CO₂. To evaluate the effects of addition of the hyperpolarized ¹³C pyruvic acid on the cells, pyruvic acid at a concentration of 100 mM was prepared in the respective medium. Cell medium was discarded and replaced with the pyruvic acid containing medium. Supernatant was collected 30s after addition of the pyruvic acid containing medium. The amount of extracellular lactate was determined using a commercially available assay (Lactate Assay Kit, Sigma-Aldrich), which uses an enzymatic conversion that results in a colorimetric product that is proportional to the lactate in the sample. Preparation of the analysis was done according to the manufacturers' instructions and extinction was measured with an ELISA reader at 570 nm (Bio-Tek ELISA Reader EL 800, Bio-Tek Instruments GmbH, Bad Friedrichshall, Germany). Experiments were carried out in triplicates. The lactate concentration in the samples was calculated based on a standard curve of known lactate samples and is displayed in ng/µl.