Proliferation Assays
Most chemotherapeutic agents inhibit oncogenesis by reducing the rate of proliferation, usually as a result of cell cycle arrest. Cell proliferation is commonly measured through an ELISA-based approach called BrdU assay. Its working principle involves the incorporation of BrdU in actively proliferating cells. The amount of incorporated BrdU is detected by incubating with anti-BrdU antibody following fixing, permeabilization and DNA denaturation. Horseradish peroxidase-conjugated antibodies bind to the primary antibody and this catalyses TMB to a blue substrate which could be measured by absorbance.
With a reduction in proliferation, cells could undergo senescence in response to a treatment. Increased activity of β-galactosidase is a marker in senescent cells. The CellEvent Senescence Green Detection Kit employs β-galactosidase’s enzymatic activity with which hydrolytic cleavage of fluorescein-based substrate leads to emission of a fluorescent signal. Positively stained cells specify the proportion of cells that have entered cellular senescence.
BrdU Assay
HLF (human lung fibroblasts) cells were seeded at an optimised density and incubated overnight at 37oC in a humidified incubator. The cells were treated with PDGF-BB and Mitomycin C. The BrdU assay showed that treatment with PDGF-BB and 10% FBS resulted in a significant increase in the cell proliferation, as indicated by the increase in the absorbance values. Treatment with Mitomycin C significantly reduced the absorbance, suggesting an inhibition in cell proliferation (***p<0.001 ± SEM).
MTT
HMC1.2 cells were treated with the test compound at 6 different concentrations. The % cell viability post-compound-treatment was determined by means of a MTT assay. A significant reduction in cell viability was observed with δ-Tocopherol when compared to the positive and vehicle controls (***p<0.001; n=5; ±SEM)
Alamar Blue
Human umbilical vein endotheial cells (HUVEC) were seeded at different densities and the proliferation was recorded over 24 hours as % signal of Alamar Blue dye.
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