I use the following equation to calculate:
Percentage difference between treated and control cells
=
(O2 x A1) - (O1 x A2)
(O2 x P1) - (O1 x P2)
x100
Where:
- O1 = molar extinction coefficient (E) of oxidized alamarBlue (blue) at 570 nm
- O2 = E of oxidized alamarBlue at 600 nm
- A1 = absorbance of test wells at 570 nm
- A2 = absorbance of test wells at 600 nm
- P1 = absorbance of positive growth control well (cells plus alamarBlue but no test agent) at 570 nm
- P2 = absorbance of positive growth control well (cells plus alamarBlue but no test agent) at 600 nm
First question: Is this equation okay for the resazurin test?
Second question: My results show that P1 and P2 are equal or less than the A1 and A2. The color of positive control(P1 and P2) becomes bright pink. I think the control absorbance (suspension of cells without drugs + resazurin) should always be more than the group of cells treated with drugs to calculate correctly. While the absorbance of my control is less than the samples. I checked the resazurin and the ELISA reader and I am sure of the authenticity of them.
What do you think is the problem?
Thanks.
Yes, the equation you have provided is correct for calculating the percentage difference in reduction of resazurin between treated and control cells in the resazurin assay. This is a standard way to quantify the results.
Regarding your second question - you are absolutely right that the positive control wells (cells + resazurin but no drug) should show more metabolic activity and reduce more resazurin (thus give higher absorbance readings) compared to the drug-treated wells.
Some things to troubleshoot:
- Make sure the positive control cells are healthy and actively proliferating/metabolizing. Old, dense or contaminated cultures could give low readings.
- Use the optimal cell seeding density. Too low density can reduce metabolic activity.
- Allow sufficient incubation time for the cells to reduce resazurin before measuring absorbance.
- There could be an issue with resazurin stock solution if the positive control wells turn brightly pink. The conversion to fluorescent resorufin indicates reduction, but there should still be enough oxidized resazurin left to allow quantification. Make fresh stock.
- carefully confirm there are no pipetting errors mixing cells, drugs, resazurin etc.
I would first repeat the assay with fresh optimized positive control cells + new resazurin to see if gives expected higher readings compared to treated cells.
Your explanation is very useful and valuable.
Thank you for your time.
Thank you very much.
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