I have done MTT assay, RTCA and finally determined IC50 of a compound. However, during apoptotic study, the result did not show 50% of cells were dead or apoptotic. It was less than 50% and it was just different 10% to 18% more apoptotic and dead cells compared to non-treated cells. It this normal or if not, Why it is so?
in MTT, normally we seeded 25,000 cells per well per 100 ul. However, when we do apoptosis analysis (by flowcytometery) we have to seed 500,000 to 800,000 cells per well per 2 ml in each well of 6 well plate. I used the same IC50 final concentration of the compound. However, I did not consider the factor of number of cells present in the well.
Do we need to increase concentration of compound higher than IC50 because of number of cells are much higher than the MTT assays in 96 wells plate which gave IC50.
If we need to increase compound concentration beyond the IC50 for apoptotic study according to the number of cells, how should we calculate to have appropriate concentration the compound based on IC50 in order to show apoptotic and dead cell to be around 50% compare to non-treated control cells.
I did annexin V apoptosis and dead cells study with flowcytometery. With the same IC50 concentration of the compound to study apoptosis, under microscope, it seems cells were noticeably inhibited growth and also seemed to be higher apoptotic or dead. However, when we check with annexin V with flowcytometry, as mentioned, it was just different 10 to 18% only. Is this because most of nearly dead and late apoptotic cells were fragmented during harvesting, staining with annexin dye that caused number of apoptotic and dead cells reduced much and left only live cells in treated sample? I cannot think why it is so. Appreciate if you could share your experience. Thanks.
It could be possible that the treatment is slowing the growth of cells so that the MTT assay reads out a 50% reduction in cell number, but the number is not due to 50% cell death, but instead you are seeing the difference caused by slowed growth and reduced cell number. You May have to raise the treatment dose to see 50% death as detected by Annexin V.
Yes, I agree with you. This could be one of the reasons too. IC =Inhibitory Concentration.
So for annexin V study, Is that normal that we are supposed to increase concentration of the compound beyond IC50? If yes, how to calculate to know how much to increase. Thanks.
Hi Jamail, I guess I am in the same boat as you. My IC50 results do not tally with my 7-AAD results, single staining for now. It seems the flow results has lower cell death compared to what was expected. It may be as what has been explained by Andrew that the IC50 from MTS/viability assay cannot differentiate slow growing treated cells/ inhibited cells that might still be viable (not actively metabolic but flow results might still show that the cells have membrane integrity intact thus they are considered as live cells? Thus low viability assay signal but high live/viability cell count in flow = low IC50. Just maybe I guess) within the incubation period. But this phenomena happened when I used a HDAC inhibitor which might reactivate various genes while it was opposite for mitotic treatment where it remained 50% cell death at 0.25xIC50 up until 1xIC50 (probably mitotic catastrophe or slow growth-> cells may remain their slow metabolic activity hence giving high IC50 in viability assay but 0.25x actually managed to kill 50% cells based on flow). I will be running PI staining to see what stage the cells are in when they are treated. However, it will be quite problematic when flow results do not tally with MTS/viability results especially when HDAC inhibitor causes various gene activation/reactivation.
Reagan Entigu, I have solved my issue. I took a 3 to 4 days break and think, browse web, and checked back all mine and finally I noted that I wrongly took old dissolved compound instead of fresh. My compound is stable for about one month after dissolve it in DMSO. However, as I did not throw the old ones, this made me picked the old one instead. With the new compound, it works and reflected 50% dead and/or apoptotic and tally with my MTT result too. I seeded 250,000 cells per ml for 2 ml each well in 6 well plate (total 500,000 cells per 2ml per well)-24 hrs incubate and at 48 hrs from seeding day, I treated the cell with IC50 of my compound. Pls note MTT/MTS is 100 ul and now we are using 2 ml media in 6 well-plate. Calculate IC50 base on 2 ml and treat them. Hopefully you will get the expected results. Cheers!
Pls check back and do again. See how your results. If any, let me know.
Hi Jamail, there are few things I can do I guess. 1st is to test IC50 on 200k cells/ml and see if it translates to flow 50% dead, because my IC50 is a scale up from 96 well plate 10k cells. Another things is I have only done 7aad which can only differentiate live and dead. Early apoptotic and growth arrested cell is also considered viable if the membrane integrity remain intact. These might explain the discrepancy between MTS and 7aad. I will be running PI staining, to determine the stage of cell cycle the cells are in. There is also that MTS signal increased for a while in the apoptotic process which also cna be looked at.
IC 50 meant Inhibitory. Not necessary to be killed exactly 50%. This meant counted for dead, late or early apoptotic cells too as long as cell are not functioning well.
I would like to check whether anyone calculate for GI50 too?
Jamail, this mean your scale up is accurate? Meaning your scale up is linear? How do you determine IC50 scale up factor? For example if 10000 cells having 1uM IC50, does this mean the IC50 remain 1uM? I need some calculation to properly scale up to get the same effect. Could you advice me on this?
For my compound, it doesn't really matter on number of cells. As long as you keep IC50 value accordingly as per volume of media, it works. This is consistent with positive control compound cisplatin that I used too. This make sense about IC50 in common sense and this should be the that way.
On the other hand, I had heard it should be depending on the number of cells. As you raised up. If one compound IC50 is 1 uM for 10000 cells, when you use 100,000 cells, it seems to increase 10 times of IC50 value of 10,000 cells. For me this does not make sense at first when I heard about this. Why? for example, you can treat 10,000 cells per well per 100 ul in 96 well. Make it 10 wells. Treat separately each well of 1 uM IC50. 24 hrs after treating, you pool back all in one large well. So in this large well, you have now total 100,000 cells in media 1 ml, however, concentration of your compound is still 1 uM too.
I believe that all compounds might have their limited range of cells numbers on which IC50 still shows the same effect. Just my thought but might be wrong. However, for cisplatin, no matter how many cells, its IC50 is still the same. This might be ideal compound.
Yes, that was what I heard. I do not want to waste more time retesting viability. Going to be a great challenge getting it right because I have various cell lines and more than 10 anticancer drugs to retest their IC50 if I have to test IC50 at another cell seeding. Ultimately my project will be looking into possible pathways that drug combinations can greatly affect. I guess I need to run PI which will offer in depth info on the cells cycle after treatments. Thanks for the feeback though.
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