We add resazurine to the following conditions:
Just media (neg Control)
Non-transfected cells (our positive control)
Non-transfected cells + drug
Transfected cells
Transfected cells + drug
To calculate the cell viability using Alamar blue we are measuring spectrophotometry at 570nm and 600nm
We are using the following formula to obtain % cell viability relative to positive control cells:
100x ((ΔAbs sample)-(ΔAbs neg CT)) / ((ΔAbs pos CT)-(ΔAbs neg CT)),
and ΔAbs = Abs(570nm) - Abs(600nm)
My rational is that:
If (Pos CT-Neg CT) -------- 100% viability
Than (Condition-Neg CT)----- x
Is that to simplistic?
Because I have read this info:
http://www.abdserotec.com/measuring-cytotoxicity-proliferation-spectrophotometry-fluorescence-alamarblue.html
and I indeed can find a formula that looks alike this one but only for fluorescence.
Should we read fluorescence instead to calculate the % of viability relative to control cells?
Do we really need to introduce the values of the Extinction coefficient of the reduced/oxidized form of resazurine in both wavelenghts?
Thanks in advance
Hi Pedro,
Q-Should we read fluorescence instead to calculate the % of viability relative to control cells?
Measuring Abs with Alamar Blue (AB) does not give you the specificity that fluorescence does, an easy way to check this is to seed cells at a range of densities and check the R^2 of the line of best fit for fluorescence (550/590nm) vs cell number. If you haven't already you should also check the % AB you are using, I would suggest checking a range between 0-40% and the length of time you leave the AB @ 37 degrees, e.g. between 2-5 hrs. Another hint is to leave your plates @RT 15 minutes after removing from the incubator before reading fluorescence to reduce edge effects.
Q-most correct formula to assess cell viability with Alamar Blue assay
First off what are you resuspending your drug in? Commonly DMSO is used, and the same final % DMSO your drug is resuspended in should be used in your neg control, or at least you should check that this final % DMSO does not effect your cells.
With Fluorescence I use: normalised cell viability= (sample RFU - pos control RFU) / (neg control (DMSO) RFU - pos control RFU)
The above formula relies on the positive control killing the cells e.g. to 10-20% and the negative control having no effect e.g. 100%. If you don't have a positive control which is harming the cells, then I would remove it from the formula and just use: sample RFU / neg control RFU
Hope this helps
Henry Beetham
Hi Pedro,
Q-Should we read fluorescence instead to calculate the % of viability relative to control cells?
Measuring Abs with Alamar Blue (AB) does not give you the specificity that fluorescence does, an easy way to check this is to seed cells at a range of densities and check the R^2 of the line of best fit for fluorescence (550/590nm) vs cell number. If you haven't already you should also check the % AB you are using, I would suggest checking a range between 0-40% and the length of time you leave the AB @ 37 degrees, e.g. between 2-5 hrs. Another hint is to leave your plates @RT 15 minutes after removing from the incubator before reading fluorescence to reduce edge effects.
Q-most correct formula to assess cell viability with Alamar Blue assay
First off what are you resuspending your drug in? Commonly DMSO is used, and the same final % DMSO your drug is resuspended in should be used in your neg control, or at least you should check that this final % DMSO does not effect your cells.
With Fluorescence I use: normalised cell viability= (sample RFU - pos control RFU) / (neg control (DMSO) RFU - pos control RFU)
The above formula relies on the positive control killing the cells e.g. to 10-20% and the negative control having no effect e.g. 100%. If you don't have a positive control which is harming the cells, then I would remove it from the formula and just use: sample RFU / neg control RFU
Hope this helps
Henry Beetham
Hi Pedro,
I am totally agree with Henry, if you measure fluorescence instead of absorbance you will have more reliable results, and I will add the solvent of your drug to your cells in order to have a reliable negative control.
In addition, I will suggest you that before adding the Alamar Blue reagent you exchange the culture medium by fresh one + % Alamar blue that you consider (I used to add 10%, but it will be better if you do a dose-dependent assay, since that will depend on your cell line basal metabolic activity). This is because the wasted components from cells present in the culture medium could also reduce resazurin to resofurin leading to a possible overstimation of your final fluorescence value and therefore an overstimation of your cell number/viability.
I hope this also helps.
Best regards,
Rodrigo
Dear Pedro,
You should exchange the culture medium with freshly prepared alamar blue, assay dose, depending on the cell line solution. This for the purposes of assessing the overestimation of the final value of fluorescence associated with cell number / viability. best regards
Thank you everyone for your aswers, they were really helpful!
We indeed always add the resazurine to a new culture medium.
For now on we will start to use fluorescence instead and use a formula that divides the RFU (condition) by the RFU(positive control, cell with no drug, non-transfected)
Best wishes,
Pedro
Hi I have another question,
How do I represent my results if the values of viability for my treatment are higher than the control?
In brief, my treatment is making the cells proliferate more than the control therefore I end up with viability over 100%. What am I supposed to do?
Thanks in advance
Edo
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