The overall goal of this method is to use isotope tracing to measure the metabolism of sorted populations of complex cell communities. So with this method, we can study the metabolic differences between subpopulations of complex cell communities, and also the metabolic collaboration between those subpopulations. The main advantage with this technique is that by combining isotope tracing with cell sorting, we can get a more reliable measure of metabolism, and also we can validate how reliable the method is.
Visual demonstration of this method is critical, as it combine different techniques, and the way cells are handled and the length of the procedure might affect cell metabolism. For metabolite extraction from a dish, first culture cells in a six-well plate in stable isotope labeled culture medium containing dialyzed supplements. When the cells reach 75%confluency, rinse the wells two times with 500 microliters of cold HBSS containing uniformly labeled glucose.
Then add 600 microliters of 100%dry ice precooled methanol to each well, and transfer the plate to the dry ice. Use a cell scraper to detach the cells and then carefully transfer the extracts from each well into individual microcentrifuge tubes. Store cells at minus 80 degrees Celsius until mass spectrometry analysis.
For metabolite extraction of sorted cells, culture cells in a 100 millimeter dish in labeled culture medium with dialyzed supplements, so that they reach 75%confluency by the sorting day. For pulse labeling of cell cycle sorted cells, culture the cells in unlabeled medium containing dialyzed supplements until they reach 75%confluency. Then, pulse label the cells for two hours by replacing the medium with fresh labeled culture medium.
Rinse the dish with warm HBSS containing uniformly labeled glucose, and detach the cells with 1.5 milliliters of trypsin EDTA at 37 degrees Celsius. After four minutes, deactivate the trypsin with three milliliters of ice cold HBSS containing labeled glucose and dialyzed supplements, and transfer the cells to a 15 milliliter conical tube for centrifugation. Resuspend the pellet in HBSS containing labeled glucose, dialyzed supplements, and EDTA at a one to two times 10 to the six cells per milliliter concentration, and filter the cells through a 40-micron strainer to obtain a single cell suspension.
Transfer the cells into a five milliliter tube and sort them on a flow cytometer. For metabolite extraction from mock sorted cells, gait out the debris and sort only the singlets. For metabolite extraction from cell cycle sorted cells, gait out the debris and doublets, and sort cells into G1 and SG2M, based on the geminin probe fluorescence.
At the end of the sort, collect the cells by centrifugation and resuspend the pellet in 50 microliters of ice cold distilled water to obtain a homogeneous pellet. Then quickly extract the metabolites by adding 540 microliters of dry ice cooled methanol. Store the sample at minus 80 degrees Celsius until LCMS analysis.
The extraction of metabolites from sorted cells must be well planned in advance, with experimental work being completed as quickly as possible to minimize the metabolic alterations. For analysis of the extracted metabolites, first select a number of metabolites with corresponding available standards that exhibit good quality sample peaks. Next, verify the quality of the peaks, taking care not to include false isotopes.
Calculate the mass isotope from our fractions for the isotope labeled samples by dividing the peak area of each mass isotope from our fraction by the total peak areas of all the mass isotopes from our fractions. Then, calculate the labeled carbon fractions for the enrichment of carbon-13 using the formula for carbon enrichment. Finally, calculate the labeled nitrogen fractions for the enrichment of nitrogen-15 using the formula for nitrogen enrichment.
In this representative experiment, 66 metabolites selected from the data analysis were present in the mock sorted cells, 60 of which appeared to be labeled with similar mass isotope from our distributions between the dish extracts and the mock sorted cells. For example, the mass isotope from our distribution of glutamate is similar between the dish and mock sorted extracts, indicating that the glutamate mass isotope from our distribution is also reliable in sorted cell populations. Analysis of HELA cells sorted into either G0, G1, or SG2M cell cycle stages using the FUCCI geminin probe reveals cytidine labeling in both populations with a higher enrichment in the SG2M stage, consistent with an increased de novo synthesis of nucleotides during the S phase.
In this experiment, the mass isotope from our distribution exhibited the same pattern in both of the cell cycle sorted fractions, suggesting that the same synthesis pathway is used, but is more active in the SG2M stage, demonstrating that metabolic differences are readily detectable by this method, even between closely related subpopulations. After watching this video, you should have a good grasp on how to extract metabolites from sorted fractions, and understand the caveats. While attempting this procedure, it's important to remember to handle the cells quickly during sorting and metabolite extraction, as the manner and the length of time cells are handled can affect their metabolism.
Once mastered, this technique can be performed in five hours. This procedure can be applied to other cell types. For example, immune cell subsets in blood, or different types of cells within a tumor, using a variety of probes, antibodies, or staining methods for sorting.
