This might be a very basic question, but I am looking to obtain a clear solution to a problem that I am encountering while performing MTT assays.
So, my experimental layout is like this:
Blank cells - The outermost cells (periphery) in my 96 well plate is made up of media only
Control cell - I have control cells comprising of cells+media only
Test cells - I have sextuplicates setup per drug concentration - with 9 different concentrations, excluding 0 concentration. These cells contain cells+drug in DMSO vehicle+media
Vehicle control cell - I have individual vehicle control cells, where DMSO is added at exactly same concentration as the drug. So I have 9 different concentrations of DMSO as well. These cells contain cells+DMSO+media.
The cells are seeded on Day 0. Drugs are added on Day1 after changing media. MTT assay is performed on Day4 and solubilization is performed using DMSO.
The problem that I encounter is - DMSO is less toxic on my cells as compared to the drug being used. Hence I get a relatively high absorbance for my vehicle control cells as compared to my test cells. Subsequently, I get negative values for my test samples, if I attempt to control them with the vehicle control. I hence needed to understand very clearly - (a) how do I effect the impact of vehicle control on my cells relative to the drug treated cells? (b) Please provide clear calculations for my understanding (c) If you find my entire procedure/part of the procedure to be faulty, please recommend changes.
Hello Deepak,
Based on the solubility of your drug, you can either choose to go with the culture media as your vehicle control i.e (media+cells) or a solvent like DMSO in your vehicle i.e (media+DMSO+cells).
What is the percentage of DMSO you use in your vehicle? It shouldn't be more than 1% in preparations involving cells. Ideally
Hi Jaison,
Thanks for your answer. The DMSO percentage used in my cells is quite low - so I am not really concerned about that. You have however, not answered my query. I am intending to understand as to how do I take the impact of vehicle control in my final calculations? Ideally it is recommended to subtract the absorbance obtained for vehicle control from the test wells. But since the vehicle control does not cause higher toxicity as compared to the drug - this calculation would yield negative values.
I am trying to understand how do I go about this issue.
Thanks again.
It sounds like your protocol is just a bit confused. Let's take a super simple system of three conditions: (A) has cells and media. (B) has cells and media and DMSO. (C) has cells and media and DMSO and drug.
MTT assay creates a purple color, and the more living cells, the more purple. We'll call your readout P. So you have three values: P_A, P_B, and P_C for the amount of purple measured in each sample.
To find the effects of the DMSO, which we'll call P_DMSO, you want P_A - P_B. This is giving you the amount of purple that was created just by the cells DMSO alone didn't kill. You could then add that to P_C to get P_C_-DMSO, or the amount of purple you'd expect in C if you hadn't had any DMSO around.
All that being said: this seems needlessly complex. I'd ignore the no DMSO sample: it's pointless, since you have DMSO in all your drug samples. So just use P_B as your 100% Growth standard. There's no need to correct anything, and so no negative values.
THAT being said: negative values don't actually matter. At all. If you want to do whatever adjustments you have, go for it. Then just scale the values to percent inhibition. The equation for this is:
( (Value_For_Maximum_Growth - Value_For_Minimum_Growth) - (Value_for_This_Condition - Value_For_Minimum_Growth)) / (Value_For_Maximum_Growth - Value_For_Minimum_Growth)
or ( (Max - Min) - (Actual - Min) ) / (Max - Min)
Where Max/Maximum Growth is the reading for your 0 drug control, Min/Minimum Growth is the reading for your starting number of cells (or your media-only controls, the way you have the experiment set up, would be close enough probably), and Actual/This_Condition is the reading for the actual sample that you're scaling. This will give you all numbers between 0 and 1, with 100% inhibition giving a reading of 1 and 0% inhibition giving a reading of 0. Just subtract the numbers from 1 if you'd rather look at %growth than %inhibition. And negative values won't matter at all in this sort of scaling analysis.
Hi Deepak,
I agree with william, but I think that your protocol is perfect.
The blank cell (no cell in the well, just media) is essential to determine your background. The OD obtain in your blank cell must be subtract from ALL your other values.
To determine the impact of the vehicle (DMSO) on your cells. You have to do a T.test between your control cells (cell+media) and your vehicule control cells (cell+media+ equivalent in DMSO). If there is any difference, like william said, the control cells (cell+media) are then useless. If you add biological replicate, you can avoid to test the control cells.
So, to dertemine the effect of your drug, you will have to compare your vehicule control cells (cell+DMSO+media), and your test cells (cell+media+drug).
One missing control in your protocol ; your positive control of death cells. Add 10% DMSO in a well. You should be able to read only your background OD.
Hope this help. Good luck.
Thanks a lot William and Hermance for the fantastic inputs!
Indeed this will help me a lot!
- Badges
- Science topic
- Similar topics
- Philosophy
- Esthetics
- Interpretation