For quality results we recomend at least six biological replicates for each condition, for example six cell culture samples treated with drug X and six cell culture samples treated with DMSO.
We recommend 6 million cells minimal per analytical analysis. For instance for each GC-MS replicate it will take one 10 cm plate worth of cells. This is also true for LC-MS. For all analysis we recommend 6 biological replicates for each condition.
Once cell culture has come to desired confluence remove media. Wash plate with PBS buffer then remove as much PBS as possible. At this stage you have several options.
Option 1) If you can remove the cells from the plate by trypsin or other method, transfer the cell culture to a microfuge tube, pellet by centrifugation then remove as much supernatant as possible. Snap freeze on dry ice and send us the sample via FedEx on dry ice. This is the preferred method. We will add several internal standards to the extraction for data normalization.
Option 2) Extract the cells yourself. After PBS add 1 mL of 80% MeOH solution to the plate (-20C), scrape cells into a microfuge tube, vortex and incubate for 1 hour at -20C. Centrifuge for 5 minutes, 4C at highest speed. Remove supernatant to new tube. Dry supernatant in a speed vac. Send us the samples in a zip lock bag with small desiccant bags added.
Collect serum or plasma per usual. The only caveat is do not use sodium citrate in the preparation of samples. This strongly affects the results. Once you have serum collected snap freeze 20-50 uL of it for metabolomics analysis and bring to the Core. This is the preferred method of sample submission.
If you wish to extract the metabolites from serum/plasma follow these instructions. To 50 uL of serum add 450 uL of solution 90% methanol (-20C) and vortex the mixture for 10 seconds followed by 1 hour incubation at -20C. Centrifuge the mixture at 14,000 rpm for 5 min. @ 4˚C to precipitate the protein. Transfer the supernatant containing the metabolites to a fresh tube and dry this en vacuo using a Speed-Vac. Perform the extraction on ice or in an -20C type enzyme caddy.
For a single analysis (one GC-MS or one LC-MS) 5 total OD’s of cells are needed. For example for cells harvested at a 1 OD, 5 milliliters of this culture will be needed. For metabolite profiling pellet 5 OD’s of cells in a 15 mL conical tube, remove supernatant and snap freeze. Drop these samples off at the Core. This is the preferred method.
If you wish to extract the cultures follow this procedure. Heat up a water bath to 80C. Make a 75% ethanol solution diluting with water. Heat this solution in the water bath carefully. Once hot add 5 mL of this solution to each sample in the 15 mL tube, mix well by vortex and then incubate in the hot water bath for 5 minutes. Post incubation pellet the cell debris, transfer the supernatant to a fresh tube and completly dry using a Speed-Vac.
We prefer to prepare all tissue samples in the Core. We use a high speed bead mill for this. Tissue preperation is sample dependent, please contact the Director for a discussion on methods.
