Multiskan rc plate reader

The MTT (Methyl thiazolyl diphenyl-tetrazolium bromide) reduction assay combines the measurement of cell proliferation with the assessment of mitochondria activity. Mitochondrial dehydrogenases of only viable cells will convert MTT to formazan products within the mitochondria. First, 20,000 cells/well were cultured in triplicates in a 96-well plate and incubated overnight. Cells were incubated at 21 or 0.2% O₂ with or without (0.05, 0.1 and 0.3 uM) doxorubicin (Dox) for 72 h. Then, 500 mM of freshly prepared MTT reagent (Sigma Aldrich, St. Louis, MI, USA, #M5655) was added to each well and incubated for 4 h at 37 °C in the dark. The medium was removed and 100µL of dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MI, USA) solvent was added to dissolve the purple precipitate of formazan. Absorbance was measured at 570 nm (reference wavelength is 620 nm) by a Multiskan RC plate reader (Thermo Labsystems, Helsinki, Finland).

Cells were plated in a 6-well culture dish (50,000 cells per well). Viable cells were stained 24h later using 0.5mg/ml MTT for 15 min (Sigma). Cells were washed with PBS and then dissolved using 0.5ml DMSO. The absorbance of the solution was measured at 590nm using Multiskan RC plate reader (Thermo Scientific).

The BrdU assay was performed using the Cell Proliferation ELISA BrdU assay (Roche, Basel, Switzerland, #11647229001) according to the manufacturer. Briefly, 20,000 cells/well were cultured in triplicate in 96-well plates using a complete culture medium and incubated for 72 h at 21% normoxic or 0.2% O₂ hypoxic environments. After incubation, 10 mM of BrdU labeling solution was added, and cells were incubated for 4.5 h at 37 °C. The labeling medium was removed, and cells were fixed, followed by staining with an anti-BrdU-POD working solution for 90 min at room temperature. Wells were rinsed three times with 1X PBS followed by color development by adding substrate and 1 M H₂SO₄ stop solution. Photometric detection was carried out within 5 min of adding stop solution at 450 nm (reference wavelength is 690 nm) by Multiskan RC plate reader (Thermo Labsystems, Helsinki, Finland).