Hi Yosef Levi. It is reasonable that the cells give lower absorbance readings for MTT after serum starvation as MTT assay measures cell viability based on the metabolic activity of the cells (ability of the cells' enzymes to convert MTT into insoluble formazan product) and serum starvation will lower the basal metabolic activity by suppressing cell proliferation and even possible to induce apoptosis.

Since, as you said, the absorbance readings for another group treated with a cytokine are so much higher (>50%), I guess the cytokine could possibly stimulate cell proliferation as well in addition to your assumption that it inhibits apoptosis.

I'm curious to know the cytokine that you used to stimulate your cells.

Hi Yosef Levi. It is reasonable that the cells give lower absorbance readings for MTT after serum starvation as MTT assay measures cell viability based on the metabolic activity of the cells (ability of the cells' enzymes to convert MTT into insoluble formazan product) and serum starvation will lower the basal metabolic activity by suppressing cell proliferation and even possible to induce apoptosis.

Since, as you said, the absorbance readings for another group treated with a cytokine are so much higher (>50%), I guess the cytokine could possibly stimulate cell proliferation as well in addition to your assumption that it inhibits apoptosis.

I'm curious to know the cytokine that you used to stimulate your cells.

Thank you for your answer, Kong Yen Liew I used IL-1a.

Do you think a shorter incubation period or a shorter starvation period would allow me to get a better comparison?

Hello Yosef Levi

Serum starvation for 24 hours induces G0/G1 cell cycle arrest. It causes cellular stress and affects the mitochondria impacting cellular energy metabolism which is why you get a drop in MTT readouts. It also induces apoptosis in the cells. The effect of serum starvation differs for different cell lines. There are some primary cultures which cannot withstand starvation for more than 2-3 hours.

I would suggest that you add 1% or 2% serum just to maintain the health of the cells in culture. But do not alter the starvation period which is of 24 hours.

The other group of cells treated with cytokine could be nullifying the effect of serum starvation in some way either by inhibiting apoptosis as you mentioned or stimulating cellular proliferation as said by Kong Yen Liew to a little extent.

Thank you.

I guess you are trying to look at the effect of IL-1a on the cells? On apoptosis? Or on other parameters?

Since you serum starved the cells for the same period of time for both your control group and IL-1a group, the difference you saw between the two groups will tell you the effect of IL-1a on cell viability and shortening the starvation period wouldn't make a huge difference, in my opinion.

But I agree with Malcolm that serum starvation doesn't suit every cell types. Some cells may be terribly affected by serum starvation. So you have to make sure that the cells are in healthy conditions before you can conclude on the effect of IL-1a on the cells.

Serum plays a role as a carrier of exogenous and endogenous substances enabling various processes of metabolism to take place, cells need various kinds of substances for example: amino acids, proteins, lipids, carbohydrates, nucleotides, coenzymes and minerals and cofactors, these substances are often selectively transported into the cell to enable certain processes to take place, and metabolites are taken out of cells into serum. Therefore without a medium such as serum there are minimal life processes that are able to go on in cells, when cells are deprived if serum they undergo apoptosis.