Cell proliferation was measured by a Cell Proliferation ELISA BrdU kit (Roche, Germany) according to the manufacturer's protocol. Cells were seeded in a 96-well plate (5000 cells/well) and cultured for 24 h. After starvation for another 24 h in serum-free medium, cells were then incubated in the same medium supplemented with different [Ca2+]o in the presence or absence of various inhibitors for 24 h. Eight hours before the end of incubation, BrdU was added to the medium, and cells then were continually incubated for 8 hours. The absorbance at 450 nm (reference wavelength 630 nm) was measured with a scanning multi-well spectrophotometer (Amersham Pharmacia Biotech). The absorbance values correlate directly to the amount of DNA synthesis and therefore to the number of proliferating cells in culture. Stimulation is expressed as fold proliferation over basal growth of the control set as unity.
Scanning multi well spectrophotometer
Manufactured by Cytiva
The scanning multi-well spectrophotometer is a laboratory instrument used for measuring the absorbance or transmittance of light by samples in a multi-well plate format. The device can rapidly scan multiple samples and provide quantitative data on the optical properties of the samples.
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2 protocols using scanning multi well spectrophotometer
1
Cell Proliferation Assay with BrdU
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BrdU-Based Cell Proliferation Assay
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Cell proliferation was measured using a Cell Proliferation ELISA BrdU kit (Roche, Germany) according to the manufacturer's protocol. Cells were seeded in a 96-well plate (5000 cells/well) and cultured for 24 h. After starvation for another 12 h in serum-free medium, cells were incubated in the same medium supplemented with different treatments for 24 h. Six hours before the end of incubation, BrdU was added to the medium, and the cells were incubated for 6 h. The absorbance at 450 nm (reference wavelength: 630 nm) was measured with a scanning multi-well spectrophotometer (Amersham Pharmacia Biotech). The absorbance values correlated directly with the amount of DNA synthesis and therefore with the number of proliferating cells in culture. Stimulation was expressed as the fold proliferation over the basal growth of the control set as unity.
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