Hey everyone,
I did a resazurin assay in a 96 well plate using HEK293 cells and measured the fluorescent signal over time (every hour for the first 6 hours, at 24 and 48 h). I think the handling of the assay worked fine, but I still have problems analyzing the data.
As my samples are fluorescently labelled with a Rhodamine-derivative and therefore have a certrain self-fluorescence at the wavelength used for the assay, I incubated one well for each sample without the resazurin and measured it along with the rest. I wanted to substract the fluorecence values from this well from the ones with the resazurin as a blank sample. The problem is now though, that the one without resazurin are much higher in intensity for the first two measurement points and therefore I get negative values. Any ideas how I can fix this?
Thanks to all of you,
Christina
Hi Christina,
You can measure resazurin with absorbance instead of fluorescence. This is a less sensitive way of measuring differences but may help you with your problem.
You measure spectrophotometrically at 570 nm and the reference
wavelength at 600 nm.
Details of the equation used to calculate viability are in the attached pdf.
Hope this is helpful,
Eleanor
The resazurin is probably quenching the rhodamine fluorescence, so the simple subtraction idea doesn't work. I agree with Eleanor that a different measurement is required in this situation.
The cytotox tests worked really well with measuring absorption instead instead of fluorescence. Thanks a lot to you! Also the pdf was much apprechiated.
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