I am about to apply the LMS (lysosomal membrane stability) method on mussels and following the protocol I need HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). However, the companies in my country don't have it in stock and they need 4 weeks to get it. I need it for my experiment by the end of this month. Do you have any idea if I can use another buffer instead of HEPES? Thank you!!!
What is the pH of the buffer that you need to prepare? Technically, you can use any other component that secures a buffering capacity around the desired pH
Will you be running the neutral red assay by spectrophotometry (absorbance of accumulated dye) or by microscopy (time point to 50% of the cells to have the dye leaking from lysosome)?
If it is the first type of assay, you could use PBS (phosphate buffered saline), but with additional sodium chloride to reach a osmolarity simillar to the sea water:
500 mM NaCl , 2,7 mM KCl , 10 mM Na2HPO4 and 2 mM KH2PO4, pH adjusted to 7.4
If you will be running the second type of assay, I believe that it could be used the same buffer, the important is to have an osmolarity close to seawater where they are being maintained, once these organisms are osmoconformers.
I Hope it was of some help.
Cheers
Yeah, I thought so. However, I will be using freshwater mussels. Thanks
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