Hello!
I'm trying to determine the pH of a cell culture plate using phenol red present in the medium (MEM) and a plate reader. However, phenol red's absorbance changes depending on the pH.
Is there a protocol or a method to do the trick? I'm having trouble determining:
A) which suitable excitation/emission wavelengths to use
B) whether to use 1 or 2 wavelengths
C) which equations to use in case it was a 2 wavelength measurement
I tried constructing a standard curve using 4 pH points at ex/em 410/570 nm but I got a polynomial graph. I measured cultured plates and solved the polynomial graph's equation but the resulted pH values was drastically different (very acidic) than the pH range determined visually and using a strip test (neutral).
And if possible, a reference would be highly appreciated.
Thank you for your time.
I think that the easiest way to solve your problem would be to do the following:
1. Buy regular glass pH mini-electrode for regular pH meters.
2. Take a sample of your cell (about-50-100 microL) and dilute it with water.
3. Determine pH.
Important. that the water MUST be BIDISTILLED from the glass distiller. Any water from any resin purifiers will be contaminated with peroxides and more likely will be too acid. Do not buy water fro Sigma. Do it yourself.
Distilled water does not change pH of the solution, it only reduces the buffer capacity.
There are several reasons why I am highly critical to plate readers, except enzymatic assays. The main reason is that having low Volume to Surface ratio the content of a cell is highly vulnerable to artifacts that come from the air and water the researcher is using. In my control experiments I have observed that in some days accumulation of peroxides from air exceeds the basic rate of ROS (measured as H2O2) production by mitochondria oxidizing glutamate.
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