Extraction Protocols of Metabolic Flux from Cells (To handle 24 samples)
Preparation of Isotopic Metabolic Flux Samples
(Note: This is not part of the core service and will need to be done in the Investigator’s lab. Test samples need to be submitted to the core, before you do full experiment. (Five Million Cells; one time point; one control and one treatment to check the percent of incorporation)
Required isotopic labeled standards. For more information please visit http://www.isotope.com/. Concentrations will depend on the culture medium and cell line (Example DMEM media – 25 mM glucose or 4 mM L-glutamine).
Cambrige isotope Item Number
D-GLUCOSE (U-13C6, 99%)
L-GLUTAMINE (15N2, 98%)
L-GLUTAMINE (5-13C, 99%)
L-GLUTAMINE (1-13C, 99%)
L-GLUTAMINE (U-13C5, 99%)
L-TRYPTOPHAN (15N2, 98%)
Purchase metabolite free-medium. An example 13C glucose flux, use glucose free medium. .
Note: Isotopic tracers and metabolite free culture medium needs to be approved in advance by the Core prior to initiation of project
1. Prepare the 13C labeled growth medium with the recommended concentrations. Note: Example DMEM media containing 4 mM [U-13C5] glutamine,
2. Count the cells and seed 5 million cell per each replicate) in the 100mm x 20mm Tissue Culture Treated Dishes under Sterile conditions using biosafety cabinet and incubate the cells in a humidified incubator controlled at 5% CO2 and 370 C., allow them to attach at least 6h- Allow to attach cells to the plate.
3. After post attachment starve the cells for about (~6-12 hrs; depending on cell lines) in metabolite free medium. Remove the spent medium completely by aspiration and wash two times with 2 mL phosphate-buffered saline (PBS-1X) under biosafety cabinet.
4. Switch cells to the 13C labeled growth medium. Record the time at which the labeled medium starts to enter the incubator. Grow cells until they reach isotopic steady-state (as determined by preliminary optimization time course experiment).
5. Collect the medium at each time point then flash freeze the cells in the plate using liquid nitrogen (slowly pour the liq N2 in the dish ~ 1/5 of the plate) and store them -80C. Note: We need to have a 12C growth medium as a control.
6. Provide the frozen plates and media to the core.
Sample extractions (Performed by core)
1. Add 750 ul of ice cold (1:1 Methanol: Water) solutions in to the frozen cell culture plate.
2. Scrape cells from the plate and transfer in to glass vial
3. Sonicate cell suspensions (Settings: Amp 30% , 20-30s, 3 times)
4. Wash rotor( do you mean sonicator probe? ) with 10ml de-ion water in between each group and different time points.
5. Wash rotor ( probe) with Methanol and water separately between each sample sonication
CHCl3 extraction (caution: extraction should be performed in glass tubes instead of plastic vials)
6. Add 450 ul of ice cold CHCl3 (preserved at -20 for 10 min) to each sample solution in step 5 and vortex for 10 min at speed 8
7. Add 150 ul of ice cold water to the above each sample mixture and vortex for 2 min
8. Keep the solutions at -20C for 20-30min
9. Centrifuge all the sample solutions at 4000 rpm for 10 min @ 4C
10. Pipette out separate layers (organic and aqueous phase), combine them and use the entire supernatant.
11. Dry the supernatant solution @ 37C, 30-45 min.
12. Dissolve again in 500 ul of methanol:water (1:1) (vortex - 5min, sonicate - 5min, spin @ 5000rpm-5min) and filter to separate proteins
Protein separation by Amicon filters
13. Precondition the Amicon protein filters by prewashing and centrifuging them w/ 500 µl 50:50 Methanol: Water (15000 rpm @ 4C -20min) until the entire solution is filtered out
14. Discard the tube and keep the filter in a new tube
15. Transfer the samples from 6 A (vii) into the pre-washed filters and centrifuge it to collect the filtered samples. Add additional 100 µl 50:50 Methanol: Water to the filter and centrifuge it to collect the remaining metabolites if any.
16. Collect the filtered solution and dry them.
17. Re-suspend the samples using appropriate mobile phase.