If you perform those methods (facs, western etc) to investigate the cell death mode/mechanism etc, you had better to choose at least IC70 to make sure that majority of cells are dead at the given time point (48 h is suitable by the way). We sometimes even use IC90. IC50 may sometimes be a wrong choice because your cells could still give you a data for IC50 without any evidence of cell death. In this case, you had better not to use the term "cytotoxic effect", it is rather cytostatic effect or even antiproliferatif effect. Importantly, the molecular changes detected by Facs, western blot etc will be taking place at much earlier time than the time used for the cytotoxicity experiments (e.g. 12-24 h versus 48 h). As an extra info, any compound showing cytotoxic effect only beyond 100 micromolar is almost useless. No need to perform further experiments with it because almost ne chance for it to become a drug. As a last saying, higher than 5-10 micromolar IC50 may not be ideal for the same aim.  Hope all these info will be useful for you. Otherwise ask again (or e-mail me: [email protected]). All the best. 

Hope u have incubated the cells for 24h. you can use different concentration 10, 20 or 30 uM of drugs.

The response of the cells to the increasing concentrations of the drug is based on a log-scale of the concentration. Thus, I suggest to perform new MTT with for about log 3 concentration range. In your case, under 80uM the viability is still 65. I'll suggest ic50 ()ie 50% viability) concentration for further experiments (this is also routine approach in our lab), unless you do not want any specific experiment ie in ic10, ic90 or such. IC50 can be calculated using sigmoideal dose response function, in excel, for instance.

In case you are testing cytotoxic activity, try using IC50 and IC20 for flow cytometry and gene expression in 6, 12 and 24-hours treatments.

For cytotoxicity studies, the IC37 is sometimes used (see attached paper). The concentration for downstream experiments like western blot or RT-PCR depends on what are you looking at. You certainly don't want a toxicity of 50% or more when you study the expression of genes/proteins crucial for cell cycle/apoptosis etc.

MTT assay gives the result of cell viavbility after drug tretment and this assy can be use for the selection of drug for the further experiments. To see the effcts of drug at molecular level I would suggest use three doses 1. Noncytotoxic, 2. Moderate cytotoxic and 3. Higly toxic which could be the IC50. Molecular effects of drug can be observed at noncytotoxic doses even. So according to your table you may use 5, 20, 40 uM concentrations of your drug.

Depend of the drug that you are using and your inicial hypothesis, for example in my case i compare the effects of one drug in control and mutated cells and i should to choose the dosis that are afeecting my mutated cells without affect the controls. In your case i think that if you havent got control cells maybe is better that you use in addition other ecperiments to asses cytotoxicity for example see the subG1 cells with the facs or anexinV.

If I were you, I would determine the IC50 of the drug and use that along with a log concentration above and below the IC50.

My dear friend;  The answer you must find for yourself, because every worker works on different conditions for MTT assay and hence giving you a figure for concentration for the drug may or may not work for your conditions of assay.

MTT test was developed to determine IC50 (50 percent of cells death/viability) for medicines which are tested in it. Of course it should be possible to calculate IC50 using sigmoideal dose response function, but normally the researcher tries to find it in the middle of titrations. Titrations are vary, recommendable set is 8-12 dilutions, and  concentrations go from almost 100 percent variability to 100 percent death. Cells should be in log phase of proliferation, means 24 hours incubation after the passage in average.

If you perform those methods (facs, western etc) to investigate the cell death mode/mechanism etc, you had better to choose at least IC70 to make sure that majority of cells are dead at the given time point (48 h is suitable by the way). We sometimes even use IC90. IC50 may sometimes be a wrong choice because your cells could still give you a data for IC50 without any evidence of cell death. In this case, you had better not to use the term "cytotoxic effect", it is rather cytostatic effect or even antiproliferatif effect. Importantly, the molecular changes detected by Facs, western blot etc will be taking place at much earlier time than the time used for the cytotoxicity experiments (e.g. 12-24 h versus 48 h). As an extra info, any compound showing cytotoxic effect only beyond 100 micromolar is almost useless. No need to perform further experiments with it because almost ne chance for it to become a drug. As a last saying, higher than 5-10 micromolar IC50 may not be ideal for the same aim.  Hope all these info will be useful for you. Otherwise ask again (or e-mail me: [email protected]). All the best. 

Your concern is not clear. It depends on the goal that you want to achieve. If you want to determine the effect of the drug on a specific gene or protein during cell death, you can choose the concentrations of drug which give you 0 to 25% viability. Treat the cells for 24h and then perform RT-PCR, western blot and FACS. If you want to see if the drug induces an overexpression/up-regulation or under-expression/down-regulation of the protein/RNA then you have to choose a drug concentration which gives you 80% to 50% viability and treat the cells for days. Then perform your experiment. Hope this will help.

I believe that you can choose the concentration of the drug with a percentage of up to 25% of viable cells and then treated for 24 h and performed RT-PCR

To my understanding, you don't have sufficient data to decide what concentration will be most suitable for your downstream experiments. To do that, you need to estimate dose-response and calculate LC50 or any other concentration for a given mortality, but in this data set you lack the response in the lower concentration range and it is unclear what your control (=background mortality) is saying.

dear Salman

as Elena mentioned MTT assay is prepared to fine the IC50 dose of the drug of your interested in. and as you mentioned the viability of the cells in the highest concentration which you have tested is 65%, so I believe you ought to repeat your MTT assay with higher concentration up to 150uM (it's better to be a triplicate assay with 0-10-25-40-55-70-85-95-105-115-130-145uM) because the optimum range of your drug seems to be around 100 uM.

hope this could be helpful

best regards

Like Sobhkhizy, I think you have to retest your drug using higher concentrations, until you can find close to 100% of death. If you find it over 150uM, then you can be sure that is not useful for cytotoxic or even cytostatic effect.

To perform PCR or Western Blot, you should test IC50 and IC70, and test the total proteins amount you obtain, because if your product is apoptotic, for example, when the concentration is high, maybe you don´t have enough protein for your experiments. That also depends of the cell line and the amount of cells you use.

You can choose your test doses by either having arithmetic or geometric differences between them. Arithmetic - sequence differ by a constant. Geometric - ratio between successive terms is constant .

Eg: Arithmetic: 0, 10, 20, 30, 40, 50, etc., the difference 10 is constant.

Eg: Geometric: 0.1, 1, 10, 100, 1000 etc., the ratio 10 is constant,

Eg: Geometric: 1.953125, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125 etc., the ratio 2 is constant.

For initial screening try to cover from nM to microM. Choose at least 7 dose. After initial assessment you can narrow down.