Hi,
I have 3 cell lines viz. J774A.1, RAW264.7 and A549. I want to do a GCMS analysis for some experiment. Please suggest a suitable method for analyte extraction.
Thank you
Hi Sandeep,
it depends on the analytes you want to measure. Important steps are the quenching step, the extraction and the derivatization.
For quenching, I used ice-cold 0.9%(w/v) NaCl solution and snap freeze with liquid N2;
For extraction, I used ice-cold 50% Acetonitrile solution, followed by another snap-freeze;
For derivatization, it depends on the metabolites and your GC-MS method. Search the literature, and if you want I can point you my articles (i used MDCK and human cells).
You should also do a sensitivity analysis of your GC-MS method. Meaning to extract 0.5, 1, 2, 3, 4...million cells and check what is the minimum quantity needed to quantify the metabolites you want to measure.
Good luck!
GC-MS is used for different kind of organic compounds, volatile or not. You just need to carry out derivatization of compounds wthen these are non-volatile. Volatile compound are injected directly.
But you need to seed cell in huge quantity to get required amount of compound.
Thank you all for your suggestions. I am a GC-MS user and have accessibility to a 2D GCMS.
Daniel A.M. Pais I want to do a global metabolomics and will decide later on if i need to to targeted metabolomics based on results. Can you please elaborate this? For quenching, I used ice-cold 0.9%(w/v) NaCl solution and snap freeze with liquid N2. Did you mean after scrapping the cells, i have to snap freeze?
Hi Sandeep,
Yes, it is as you said!
I worked with adherent cells, but for suspension cells the process is similar.
Basically the quenching with ice-cold 0.9%(w/v) NaCl "stops" the metabolism of the cells. I removed cell medium and did a wash with this solution; then I dipped the 6-well plate into liquid N2, I scrapped the cells pippeted everything into an eppendorf and dipped the eppendorf into liquid N2 again, storing at -80 afterwards.
Best,
Daniel
Sorry, my mistake:
The correct protocol is
Basically the quenching with ice-cold 0.9%(w/v) NaCl "stops" the metabolism of the cells. I removed cell medium and did a wash with this solution; I removed the solution, then I dipped the 6-well plate into liquid N2: I added ice-cold 50% acetonitrile solution and I scrapped the cells, pippeting everything into an eppendorf and dipped the eppendorf into liquid N2 again, storing at -80 afterwards.
Sorry for the mistake.
Best,
Daniel
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