First, calculate the mean of your OD for each samples. You may convert  the value to % of cells viability. Example:

For control : mean OD control/mean OD control *100 =100 %

as for treatment : mean OD treatment/mean OD control *100=_%

The suggestion of Nazilah is the best...you can put this equation in Excel or use any software, GraphPad Prism or another.

IMPORTANT: Make sure to substract the background (empty wells, or in your case, solvent control) first, otherwise you will not be able to appreciate the relative changes appropriately.

Example:

Background fluorescence: 500

Sample A: 800

Control: 600

Relative Ratio (Sample/Control): 800 / 600 = 133%

True relative ratio: (Sample-Background)/(Control-Background) : (800-500)/(600-500)=300%

I also agree with Christian.....make sure to subtract background

 Thank you for all valuable information  

To clarify, I've tried to sum up my usual workflow and added an example excel sheet:

To calculate a viability assay like MTT, do the following:

1) make an average of a few "empty" wells that contain your MTT solution but *no* cells. This will serve as a background control (= "blank"). Because you will calculate percentages/ratios, you need to define what the baseline (= zero viability = no cells) is, otherwise your data is meaningless.

2) substract your background control from step 1 from all the measurements for this plate. Now your values have been corrected for background fluorescence, and you can proceed building ratios.

3) calculate an average for your control (=healthy cells with 100% viability).

4) now calulate a ratio of VALUE / control * 100 for each of your values (- background control, of course)

Thank you sir

I use medium + MTT solution as a background control

the equation will be: (OD sample-OD medium)/(OD control -OD medium) x 100%

thank you sir any reference paper for this ?

You can read protocol from Dojindo (https://www.dojindo.com/Protocol/Cell_Proliferation_Protocol_Colorimetric.pdf)

Actually it is for CCK-8, but you can use it also for MTT.

the cell survival is calculated by: (OD treated well [−blank])/(mean OD

control well [−blank]) × 100

reference:Cancer Cell Culture, Methods and Protocols,Second Edition,Edited by

Ian A. Cree

How many wells should I have for blank? Should I put the blank on the same plate as my samples or in a different one?

Please remember that the MTT assay is a surogate for cell numbers, and may not be identical to a cell number growth curve. With modern imaging capabilities, actual cell counts are just as readily obtained, in real time and can be done repeatedly on the same samples. So I would recommend using the actual datum of interest, the number of cells at a given time(s) as a function of treatment, rather than a surogate that can behave differently and confuse the picture. The precision estimates for the MTT result could be quite different from those for cell numbers and the relation between MTT and cell number then also has to be taken into account to get the true precision estimates for cell numbers. Hence the surrogate is almost always considerably less accurate.

Thank you sir

does anyone subtract the average OD value for cells with media only from the treatment wells too before calculating cell viability?

Should the cell viability (% decrease) from the control which is 100%.

Your background should be your MTT OD with media only (to be subtracted from all MTT readings in that plate or experiment). Your MTT OD readings when cells are present should represent the number of cell present. However, since cell growth is exponential (when established) and OD readings are exponential (the OD is a logarithm of light absorbance), ratios of ODs, rather than differences between them, should be taken when comparing different populations of cells (e.g., treated vs untreated). This provides a reading proportional to the number of cells, and makes more sense when precision estimates are calculated (using equipartition of variance).

Number of viable cells should increase with growth, decrease with cytotoxic treatments, and remain the same (or plateau) with cytostatic treatments.

Again, cell numbers are a more reliable measure of cell growth and survival that MTT measurements. Automated imaging methods are compatible with high throughput studies, so there is no real advantage to MTT measurements anymore (if there ever was).

First, calculate the mean of your OD for each samples. You may convert  the value to % of cells viability. Example:

For control : mean OD control/mean OD control *100 =100 %

as for treatment : mean OD treatment/mean OD control *100=_%

Hi Everyone!

Question about the standard deviation for MTT assay??

When we calculate EC50 using graphpad, we need to know standard deviation. In one column, you put % survival, in other column you put the standard deviation. How do you calculate standard deviation? Just standard deviation of absorbance of MTT formazon or standard deviation with respect to the viability. Please see the attach! Please let me know if you have thoughts about it.

Ideally, you should place your measured data (MTT ODs) in your spread sheet and then calculate the SD (or SEM if comparing means) for same condition replicates. Then use equipartition of variance to carry forward the inherited error through your conversion of MTT values to cell numbers and surviving fractions (or EC50).

What should be done if some of the values are negative after subtracting out the average no cell (media) value?

Hopefully, you have multiple readings for each condition, such that you can calculate means and standard errors (sem) for each. If so, then test for significance of the difference between the values that show a negative value difference. If not significant, then you can arbitrarily set that difference to zero, and proceed with the rest of the analysis.

Hi,

with MTT I have always 2 controls - positive and negative.

- negative has only cells + PBS treatment (as a solvent for agents being tested),

- positive has cells + control toxic compound to check dead cells optical density (Triton X100 for example). Ideally, optical density of positive control falls around blank (I use it as a brief check of running experiment).

1. find a mean for each sample

2. normalize with a blank

3. negative control as 100%

4. calculate % for each sample

@ Gardenia Pacheco

If you are investigating a toxic compound and getting negative values, check more precisely your control wells. Examine the cells under microscope, compare with other samples during each step (before adding studied toxic agents, before adding MTT, after incubation).

Control cells should look most normal, have higher amount of cells, small level of dying cells.. You can find a clue when the difference occurred.

You can also double your control. Seed a few repeats in different places in the well plate.

If you have a very high concentration of toxic agent in the surrounding wells, it can affect as well.

Hope it helps:)

Via using your standard curve and controls as reference (pos/neg).

Are you using proliferating cultures and treatment that is more than ~4 h? Then please read this. Viability assessment with proliferating cultures requires single cell analysis and cannot be done in a 96-plate format using a plate reader. I wrote the following section for the most misleading crystal violet "cytotoxicity" assay which applies to all 96-well plate "viability" assays. A major advance in cancer research will be made when such misinforming and useless assays will be removed. "The Crystal Violet "Cytotoxicity" assay with proliferating cultures will determine anything but cytotoxicity. The 2016 protocol description by Feokistova et al. is for non-proliferating cells. Please see their Fig. 1 (control wells before and after treatment; there is no change in cell number!!!). In fact, the description of all such 96-well plate assays (MTT, CellTitre-Glo, etc) for viability assessment with proliferating cultures are majorly flawed. Misinformation perpetuated by misuse of such assays, as well as large-dye uptake assays (trypan blue, PI) have been discussed (PMID 30071623 and refs therein, including PMID: 27750219). If you use the single-cell MTT assay described in PMID 30071623, you will see that cells that are assumed to be dead (even with apoptotic morphology) are viable and metabolize MTT. Like the "gold standard" colony formation assay, all 96-well plate "viability" or "cytotoxicity" assays that are performed with proliferating cultures determine the ability of a test agent to convert proliferating (dangerous) cancer cells to dormant (most dangerous, tumor repopulating) cancer cells, and not to dead cancer cells. The IC50 values measured by such assays reflects a sum of cell cycle checkpoint activation (a pro-survival mechanism), short-term dormancy due to reversible apoptosis (anastasis), and early stages of durable proliferation arrest (dormancy, due to polyploidy, multinucleation and senescence, all of which contribute to disease relapse). None of these well-documented responses is associated with loss of viability. Viability assessment with proliferating cultures requires single cell assays and cannot be determined in a multiwell plate format using a plate reader."

Hi!

I am trying to calculate the cell viability of a certain treatment.

So I have a positive control (toxic substance) and the sample.

To summarize I know the calculation after normalizing the samples with the blank would be [1-(positive control/sample)]*100 which apparently makes sense but still it is not crystal clear to me, can anyone explain this calculation?

Thank you.

you can arrange for 2 fold serial dilution and incubate Ur test plate including +ve control material and Ur test material 24 hrs decant treatment medium wash plate

Add MTT for fixed cells (viable cells) or crystal violet for methanol or formalin-fixed cells. Read using ELISA reader. Find the mean of each dilution replicates

Calculate the viability% calculated as follows ( MOD test dilution/ MOD of cell control. Find the viability% of Ur +ve control in the same way. Compare the Viability % of test sample by Ur +ve control

you can see here protocols and calculation for the MTT assay

https://cellculture.altervista.org/cell-viability-assay-mtt/?doing_wp_cron=1665641732.8867990970611572265625

thank you for the answers

The MTT assay is a method for measuring cell viability by measuring the activity of cellular enzymes. The cells are incubated with the MTT reagent, which is converted by mitochondrial enzymes into a colored formazan product. The amount of formazan product is proportional to the number of viable cells present. To calculate cell viability, the absorbance of treated cultures must be compared to the absorbance of untreated cultures. The cell viability is then calculated as a percent of the untreated control. This is done by subtracting the absorbance of the treated cultures from the absorbance of the untreated cultures, and then dividing by the absorbance of the untreated cultures. The resulting number is multiplied by 100 to give a percentage. For example, if the absorbance of the untreated cultures is 0.5 and the absorbance of the treated cultures is 0.3, the cell viability is calculated as follows: Cell viability (%) = (0.5 - 0.3) / 0.5 x 100 = 40% This indicates that the treated cultures had 40% of the viability of the untreated cultures.