Hi, I just started Seahorse experiments and I am confused with the fact that I should change the medium for assay medium 1 hour before measurements. Meaning there are changes in pH, as far as this medium doesn't contain any Hepes . Actually in my first measurement the pH is 6.3. Does someone has an idea whether it is possible to add Hepes to this medium? Will it effect O2 respiration? And in case the ph is so low, respiration will be effected anyway, wont it? Regarding the respiration whether there is any limitation in minimum respiration level? In my case it was around 20, is it reliable? Thank you!
To detect extracellular pH changes (ECAR), ideally, you need to have media with buffer capacity=0. If you are interested in O2 measurements (OCR) only then it does not matter much what media to use.
The medium is changed to remove serum (which froths in the assay) and bicarbonate and CO2 (which degass in the CO2-low atmosphere of the assay and hence alter pH), and to keep the buffering power fairly low (so you can measure ECAR) but not too low (so pH does not change too much). Adding Hepes or other non-volatile pH buffers is fine - it just means the pH changes will be smaller. And yes, pH 6.3 is rather low for most cells. We generally work at OCR >100 pmol/min/well (XF24) to ensure accuracy and reproducibility.