Hi there,
i did a little project on the side with a student in which i wanted to check, if my Fishcellline RTgillW1 can be tested at different pH Values.
So i adjusted the pH value of my Medium (L15exposure medium of OECD249) to different pH values (pH 5 to pH9) in incubated the cells for 24hs. After that i did a normal Resazurin viability assay and measured the fluorescence.
Turned out that viability in non adjusted media was 100%, in media with pH 5 and 6 viability was lower, but in pH 8 and 9 the viability increased to 130 and 140%.
Does anyone have an explanation for this? The cells looked distressed at pH 9 under the microscope... how can the values be above 100%? The media does not contain fcs, so proliferation cant be an explanation.
I also dont know how stable the adjusted pH value really was, could be that it buffered itself back again to around 7... but event if so, why is the viability higher than in the control?
Would be thankful for your input!
Hi Bjarte Skoe Erikstein , thank you for answering my question!
If i understand it right, then high pH Value could increase production of molecules which then interact with the resazurin. the high viability values are more likely "false positive" as they show cell stress, right?
In which way does Resazurin interact with ROS? More ROS = high Resazurin reaction? Or is it the other way around? Normally my cells are not showing a toxic effect from the resazurin in my controls, but in the higher pH value samples the cells looked disturbed under the microscope. so values above 140% "viability" are more likely a sign of mitochondrial stress, right?
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