In a proliferation experiment, i´ve read in many places that a positive control could be anything that "kills" all the cells (best case scenario will be the lowest concentration of your tx at which you see 100% cell death). Since we are talking about proliferation assays, wouldn´t it be better to evaluate inhibition of proliferation (besides cell death) by comparing initial cell number (positive control, 100% inhibition of proliferation) and final cell number (negative control, 0% inhibition of proliferation) vs proliferation found with your anti-proliferative drug?
In any proliferation assay, positive control mostly considered as those component that it will always kill/inhibit the cancerous cells anyway, even they are most standard market grade anti-cancer drugs.
Another condition is untreated, where NO DRUGS OR NO SUCH TREATMENT has been done.
Now, in-vitro studies of proliferation or inhibition of drug depend on cancer cell types as well as the concentration window which has used to evaluate. The concentration is always chosen is IC50.
All the time, every result won't be same even though the same conditions are being following, so we consider pattern of activity whether it is reproducing the same effect/same way or NOT.
.
lets take this two events, which are vital
I. Apoptosis is a programmed cell death that often depends on mitochondria and is irreversible.
II.“cell cycle arrest,” often called “growth arrest” can be either irreversible (“senescence”) or reversible (“return to proliferation”). This mechanisms is to skip multiple replications of DNA with error to avoid unnecessary increase in cell proliferation.
III. The p53 gene which is involved in both cell cycle arrest and apoptosis.
IV. The apoptosis pathway comprises an array of caspases regulators
Now, Do we really think that comparing cell number initial and at final stage will be enough to define proliferation rate???
Point here, proliferation assays are mostly done to get the conditional concentration window, so we can analyse the gross effect at the downstream level which will give better insight that whether effect is really affecting proliferation of not.
Regards,
Saurabh
You also need to remember, that a lot of proliferatin assays, do not measure proliferation directly but only celll viability. If you will use MT assay, then you could observe the difference in viability not due to the change in amount of cells, but also due to the change in mitochondria activity. So you need to think very carefully what your assay measures and then think if you need some additional controls or a second assay. E.g., when I got that viability of my cell culture have changed due to my chemical, then I analyzed one of concentrations with BD Accuri flow cytometer, and then I got the answer, that the amount of cells in a treated cell ulture was lower than in a non-treated control, but also cthat some cells have died, some have stopped their cell cycle only and some were still actively proliferating. So, I had changes in viability and in amount of cells, but it was due to the many factors, both cell death and cytostatic effect.
Hi Mauricio de la Rosa-Garza are you measuring cell apoptosis or cell necrosis?
Thank you all for the replies.
Saurabh Mandal I agree that comparing intial vs final cell number won´t be the best approach to determine proliferation rate, but it will be a good approach considering that i will need to carry this experiment several times and i just want a reference in order to readjust my drug concentration while developing resistance in a cancer cell line.
Patrycja Koszałka I´m currently using cyquant direct cell proliferation assay. I actually avoid MTT assays or CelltitterGlo (broadly use for proliferation assays) precisely because of what you mentioned, they measure metabolic activity rather than other component that could indicate cell number.
Steingrimur Stefansson neither. I´m measuring cell number (DNA content) to evaluate proliferation, since the drug im using is a receptor antagonist. This receptor, when activated, will normally promote cancer cell proliferation. In this context i´m determining the IC50 of my drug using several concentrations and controls. Negative control (0% inhibition of proliferation), positive control (100% inhibition of proliferation), I use these controls as "relative cell number" references in order to determine a drug concentration at which it has "no" effect on cell proliferation and another one in which it has "max" effect in inhibition of proliferation. Once i have these "min" and "max concentrations i use 4 other concentrations within this range and build a curve in order to determine the IC50 of my drug. My negative control (vehicle, DMSO) is read at the end of the experiment (72h post tx), my positive control (initial cell number, aka max inhibition of proliferation, cells didn´t undergo any kind of division during the experiment) is read 24h post seeding and before the treatment is applied to the treated wells.
Maybe my explanation is a little confusing, if you have any doubts i´ll be happy to clear them out and keep discussing about the topic.
Sorry for the bad english.
- Badges
- Science topic