Mtt dye reduction assay

Cell growth was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). Tumor cells were detached from the culture flask by enzymatic treatment using accutase (PAA Laboratories, Cölbe, Germany). Each well of a 96-well plate was then filled with 5,000 cells and exposed to a mistletoe extract or control medium without extract (each in triplicate). Cells were allowed to settle down to establish firm adhesion contact and to recover from enzymatic treatment overnight. Tumor cell number was then counted after 72 h. After 24, 48, and 72 h incubation, 10 µL MTT (0.5 mg/mL) was added for an additional 4 h. Cells were then lysed overnight in solubilization buffer (10% SDS in 0.01 M HCl) at 37 °C, 5% CO₂. A microplate reader was used to detect absorbance at 550 nm (Tecan Infinite M200, Männedorf, Switzerland). To convert the absorbance into an absolute cell number, a standard curve was prepared. In total, 2500 to 160,000 cells/well were seeded onto a 96-well plate in triplicate, subsequently lysed after metabolization of the MTT, and absorbance was measured. Data were presented graphically as dose–response kinetics, after offsetting with a standard curve and normalization to 24 h.

Cell growth was assessed using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). RCC cells (50 μl, 1 × 10⁵ cells/ml) were seeded onto 96-well tissue culture plates. After 24, 48, and 72 h MTT (0.5 mg/ml) was added for an additional 4 h. Subsequently, cells were lysed in a buffer containing 10% SDS in 0.01 M HCl. The plates were incubated overnight at 37°C, 5% CO₂. Absorbance at 550 nm was determined for each well using a microplate ELISA reader. Each experiment was done in triplicate. After subtracting background absorbance, results were expressed as 24 – 72 h cell growth rate calculated in percentage increase compared to controls set at 100%.
Cell proliferation was measured using a BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Calbiochem/Merck Biosciences, Darmstadt, Germany). Tumor cells, seeded onto 96-well microtitre plates, were incubated with 20 μl BrdU-labeling solution per well for 8 h, and then fixed and detected using anti-BrdU mAb according to the manufacturer's instructions. Absorbance was measured at 450 nm.

Cell growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). Tumor cells (100 µL, 1 × 10⁴ cells/mL) were plated into 96-well tissue culture plates. After 24, 48, and 72 h, MTT (0.5 mg/mL) was added for an additional 4 h. The reaction was stopped by lysing the cells in a buffer containing 10% SDS in 0.01 M HCl. After incubating the plates at 37 °C, 5% CO₂ overnight, the absorbance at 570 nm was measured for each well using a microplate proliferation enzyme-linked immunosorbent assay (ELISA) reader. Each experiment was performed in triplicate. After subtracting background absorbance, results were expressed as mean cell number.
Cell proliferation was measured using a BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Calbiochem/Merck Biosciences, Darmstadt, Germany). Tumor cells (50 μL, 1 × 10⁵ cells/mL), seeded onto 96-well plates, were incubated with 20 μL BrdU-labeling solution per well for 8 h, fixed and detected using anti-BrdU mAb according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate ELISA reader.

Cell growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). Tumor cells (100 µL, 1 × 10⁴ cells/mL) were plated into 96-well tissue culture plates. After 24, 48, and 72 h, MTT (0.5 mg/mL) was added for an additional 4 h. The reaction was stopped by lysing the cells in a buffer containing 10% SDS in 0.01 M HCl. After incubating the plates overnight at 37 °C and 5% CO₂, the absorbance at 570 nm was measured for each well using a microplate proliferation enzyme-linked immunosorbent assay (ELISA) reader. Each experiment was done in triplicate. After subtracting background absorbance, results were expressed as mean cell number.
Cell proliferation was measured using a BrdU cell proliferation ELISA kit (Calbiochem/Merck Biosciences, Darmstadt, Germany). Tumor cells, were seeded into 96-well tissue culture plates, incubated with 20 µL BrdU-labelling solution per well for 8 h, and fixed and detected using anti-BrdU mAb according to the manufacturer’s instructions. Absorbance was measured at 450 nm after 24 h.

Cell growth was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). Caki-1 cells (50 μl, 1 × 10⁵ cells/ml) were seeded onto 96-well tissue culture plates. After 24, 48 and 72 hrs, 10 μl MTT (0.5 mg/ml) were added for an additional 4 hrs. Thereafter, cells were lysed in a buffer containing 10% SDS in 0.01 M HCl. The plates were incubated overnight at 37°C, 5% CO₂. Absorbance at 550 nm was determined for each well using a microplate ELISA reader. A standard curve was run in parallel to calculate the cell number, assuming that mitochondrial activity was the same in all the cell cultures. Each experiment was done in triplicate. After subtracting background absorbance, results were expressed as mean cell number.

The overall metabolic activity of human hepatocytes was measured using the MTT dye reduction assay (Roche Diagnostics, Penzberg, Germany). This assay is based on the reduction of yellow tetrazolium salt (MTT) to purple formazan crystals. This only occurs in the presence of NADH or NADPH, which are only produced by metabolically active cells. The absorbance of the colored solution can be quantified by measuring a certain wavelength using a spectrophotometer.
Human hepatocytes (50μl, 5×10³ cells/ml) were seeded onto 96-well culture plates. After 24h and 48htreatment with 1000ng/ml, 100ng/ml and 10ng/ml levosimendan, cells were incubated with MTT (0.5 mg/ml) for 4h. Untreated hepatocytes served as controls, and saponin served as positive control. The cells were then lysed in a solubilisation buffer.
Subsequently, the plates were incubated at 37°C o/n. A microplate reader was used to determine the absorbance at 550 nm for each well. The wavelength was chosen according to the manufacturer’s instructions as well as in accordance with prior tests which had shown maximum absorbance in human hepatocytes at 550 nm.

Cell growth was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). Treated versus non-treated PC-3, DU-145 or LNCaP cells (100 μl, 1 × 10⁴ cells/ml) were seeded onto 96-well tissue culture plates. After 24, 48 and 72 hrs, MTT (0.5 mg/ml) was added for an additional 4 hrs. Thereafter, cells were lysed in a buffer containing 10% SDS in 0.01 M HCl. The plates were allowed to stand overnight at 37°C, 5% CO₂. Absorbance at 570 nm was determined for each well using a microplate ELISA reader. Each experiment was done in triplicate. After subtracting background absorbance, results were expressed as mean cell number.