Cell proliferation assay versus cell viability assays

Lecture 4

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 cycle

until 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).

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

Add MTT solutions and read over night

AJ-5 Anti-proliferative effect

MCF7 = 0.17 MDA-MB-231 = 0.19

DNB = 0.4631

FG0= 0.4049

CT-1= 0.4296

IC50 (µM)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

-20

0

20

40

60

80

100

120

MCF7

MDA-MB-231

FG0

DNB

CT-1

AJ-5 concentration (µM)

Cell

surv

ival

(% o

f con

trol

)

AJ-5 exerts potent anti-proliferative activities against human breast cancer cells