Cell proliferation was assessed using the commercial kits Cell Counting Kit–8 (Dojindo Molecular Technologies, Inc., Gaithersburg, MD) and DNA·IdU Labeling and Detection Kit (Takara Bio, Otsu, Japan).
The rationale for the cell counting assay using the Cell Counting Kit–8 kit is that the color-developing substrate WST–8 contained in the kit is reduced by intracellular dehydrogenase to water-soluble formazan, which can be directly quantitated photometrically. ASCs (1 × 104 cells/well) were plated in 24-well plates and incubated for 1–7 days in 1% or 20% O2. FGF–2 (1–100 ng/mL) and VEGF (50–200 ng/mL) were added to the DMEM. The absorbance was measured at 450 nm (n = 3). The spectrometry was converted into cell number. The DNA IdU Labeling and Detection Kit is a colorimetric immunoassay based on the measurement of 5-iodo–2′-deoxyuridine (IdU) incorporation during DNA synthesis. ASCs (2 × 103 cells/well) were plated in 96-well plates, incubated for 24 hours in 1% or 20% O2, and labeled with IdU. Cells were fixed, incubated with peroxidase-conjugated anti-IdU antibody, incubated with the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine, and IdU incorporation was quantitated by measuring the optical density at 450 nm. Proliferation of ASCs in 1% O2 in the presence of an inhibitor (10 μM PD98059, 10 μM LY294002, or 30 μM SB203580) was examined in a similar manner.
The rationale for the cell counting assay using the Cell Counting Kit–8 kit is that the color-developing substrate WST–8 contained in the kit is reduced by intracellular dehydrogenase to water-soluble formazan, which can be directly quantitated photometrically. ASCs (1 × 104 cells/well) were plated in 24-well plates and incubated for 1–7 days in 1% or 20% O2. FGF–2 (1–100 ng/mL) and VEGF (50–200 ng/mL) were added to the DMEM. The absorbance was measured at 450 nm (n = 3). The spectrometry was converted into cell number. The DNA IdU Labeling and Detection Kit is a colorimetric immunoassay based on the measurement of 5-iodo–2′-deoxyuridine (IdU) incorporation during DNA synthesis. ASCs (2 × 103 cells/well) were plated in 96-well plates, incubated for 24 hours in 1% or 20% O2, and labeled with IdU. Cells were fixed, incubated with peroxidase-conjugated anti-IdU antibody, incubated with the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine, and IdU incorporation was quantitated by measuring the optical density at 450 nm. Proliferation of ASCs in 1% O2 in the presence of an inhibitor (10 μM PD98059, 10 μM LY294002, or 30 μM SB203580) was examined in a similar manner.