Assays Based on Cell Proliferation • Cell counts can be used to determine the effect of various compounds on cell proliferation, but at least in the early stages of testing, a complete growth curve is required. • Growth Cycle: • After subculture, cells progress through a characteristic growth pattern of lag phase, exponential, or log phase, and stationary, or plateau phase. • population doubling time (PDT) during exponential growth • The maximum cell density achieved in the plateau phase
Why do we need PDT? • To quantify the response of the cells to different inhibitory orstimulatory culture conditions. • To monitor of the culture during serial passage and enablesthe calculation of cell yields and the dilution factor requiredat subculture.
The PDT derived from a growth curve should not beconfused with the cell cycle or generation time- what are the differences? • The cell cycle time is measured from one point in the cell cycleuntil the same point is reached again. • PDT is an average figure
PDTs vary : • 12 to 15 h in rapidly growing mouse leukemias, • 24 to 36 h in many adherent continuous cell lines • 60 or 72 h in finite cell lines. • Some cell lines have even slower rate.
Analysis of Monolayer Growth Curves • (1) Calculate the number of cells per well and cells /ml • (2) Plot the cell density (cells/cm2) and the cell concentration(cells/mL), both on a log scale, against time on a linearscale • (3) Determine the lag time, PDT, and plateau density • (4) Establish the appropriate starting density for routine passage . Repeat the growth curve at different cell concentrations if necessary • Compare growth curves under different conditions, and try to interpret the data
MTT • In cases where there are many samples, a single point in time—such as the number of cells three to five days after exposure—can be used. The time should be selected as within the log phase, and preferably mid-log phase, of control cells. • The most popular are 96-well microtitration plates or icroplates,each well having 28 to 32 mm2 of growth area, 0.1 or 0.2 mL medium, and up to 1 × 105 cells.
MTT is a yellow water-soluble tetrazolium dye that is reduced by live, but not dead, cells to a purple formazan product that is insoluble in aqueous solutions. • This cellular reduction involves the pyridine nucleotide cofactors NADH and NADPH (36). The formazan crystals formed are solubilized and the resulting colored solution is quantified using a scanning multiwell spectrophotometer (ELISA reader).
AJ-5 Anti-proliferative effect MTT Assay MCF12A,MCF7,MDA-MB-231, FG0 and DNB cells Plate cells in 96 microplates 48 hours settle Perform a dose curve of AJ-5 For 48 hours IC50 (µM) Add MTT solutions and read over night DNB = 0.4631 FG0= 0.4049 MCF7 = 0.17 CT-1= 0.4296 MDA-MB-231 = 0.19 AJ-5 exerts potent anti-proliferative activities against human breast cancer cells