I need to do some proton NMR of approx. 20-30 samples. Do you know of any good and simple ways to prepare the samples for 1H NMR (cell washing, counting, lysing, NMR solutions)? I can have approx. 10 million cells ready per sample.
Hi, all depend in which metabolites you are interested in (hydrophilic or hydrophobic). I am interested in polar metabolites and the protocol I used can be summarize in this steps:
1. Incubation--> taking sample (depending on your cells and the metabolites you are interested to see you will need more or less sample, the usual ranges from 10^5 to 10^9... you would probably need to count cells here or use a volume estimation such as one microlitre of cells has n therefore 200 microlitres will have n1)--> washes in 1X PBS (at least 2).
2.Extraction with a solvent or a combination of solvents. These solvents should preserve your metabolites. For example some metabolites are sensitive to freeze-unfreezing cycles and therefore solvents with very low freezing temperature will help. The usual ones are methanol, acetonitrile, chloroform... again all depends on your cells and what are you looking for
3.Vortexing at very low temperatures (on dry ice) with rounds of sonication if necessary.
4.Centrifugation at high speed. This is relative. There are protocols that use two centrifugation steps, one at lower speed, collect supernatant, resuspend with more extraction solution and then sonicate and centrifuge again this time at high speed or directly centrifuge at high speed (14000g) and collect supernatant.
5. Freeze -dry or nitrogen blow dry to then store at -80 until you want to read them.
6. Resuspend in D2O. Depending on your NMR tube, a different volume should be use. For example for a 5mm one you can resuspend in 600µL, vortex, spin down the tube and collect 580 that go into the NMR tube
In step 6 you can add your standard (TSP, TMS...) at a known concentration, typically 0.5 mM or as I do, add it in the extraction solution so I will go through the same losses than the metabolites in the sample.
We tested several protocols and the best one can be found in Nature Protocols, Vol.6, No.8; 2011. The title is:
Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling (by Christopher A Sellick, Rasmus Hansen, Gill M Stephens, Royston Goodacre & Alan J Dickson). Best regards