I'm currently doing Griess assay for measuring NO levels in cell supernatant for which I use the Promega kit. After collecting and centrifuging the supernatant I run the griess assay immediately. I store some aliquots of the same sample in -80C for future test. Every time I run a sample from the frozen lots, the values I get for the NO levels are not the same (frequently lower, but sometimes higher) than the freshly collected supernatant. Is this commonly experienced? Can the griess be performed on frozen samples?
First, remember that the Griess assay does not measure NO, but nitrite.
Freezing samples does not affect your ability to do the assay in any way, but it may affect the amount of nitrite in the samples. For example, some of the nitrite could oxidize to nitrate or some additional SNO or NO could oxidize to nitrite or maybe even some nitrate could be reduced to nitrite.
Are you using a nitrate reductase to measure total nitrite/nitrate in your samples? If you have to store the samples, measuring this total should give you a more stable value, though nitrite is the more biologically significant/relevant species and measuring total may mask some smaller or more subtle changes.
Finally, how quickly are you freezing the samples? Flash freeze in LN2 and storage at -80 may help. Also, you may consider using a combination of NEM/EDTA to block thiols and reduce various transition metal reactions as soon as you collect the sample. If there's a lot of protein in the samples, you could also ultrafilter them to get yield only small molecules, which should improve stability.
Ultimately, assaying immediately is always best practice when it comes to such assays, but you should be able to optimize the collection and storage conditions such that the stored samples are still useful, at least when compared to controls treated in the same fashion.
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