Hey there! I'm a supplier of Tet - 213 cells, and today I want to walk you through how to perform metabolomic screening on these cells. Metabolomic screening is a super useful technique that can help us understand the small molecule metabolites in cells, which can give us insights into cell function, disease mechanisms, and more. So, let's dive right in!
1. Preparing the Tet - 213 Cells
First things first, you gotta get your Tet - 213 cells ready. If you're getting them from me, they'll come in a cryopreserved state. Thaw the vial of cells quickly in a 37°C water bath, being careful not to let the water get into the vial. Once thawed, transfer the cells to a centrifuge tube with pre - warmed culture medium. Spin it down at a low speed, like 1000 rpm for 5 minutes, to pellet the cells.
Remove the supernatant and resuspend the cell pellet in fresh culture medium. Then, transfer the cells to a culture flask. We usually use a T25 or T75 flask depending on how many cells you have. Incubate the cells at 37°C in a humidified atmosphere with 5% CO₂. Keep an eye on them, and when they reach about 70 - 80% confluency, they're ready for the next step.
2. Sample Collection
When your Tet - 213 cells are at the right confluency, it's time to collect samples for metabolomic screening. First, aspirate the culture medium from the flask. Then, wash the cells gently with ice - cold phosphate - buffered saline (PBS) to remove any residual medium. Do this twice to make sure you get rid of all the extra stuff.
After washing, add a suitable extraction solvent to the flask. A common one is a mixture of methanol, water, and chloroform. The ratio can vary, but a 2:2:1 (methanol:water:chloroform) is often used. Scrape the cells off the flask using a cell scraper and transfer the cell - solvent mixture to a centrifuge tube. Vortex the tube vigorously for a few minutes to break up the cells and extract the metabolites.
Next, centrifuge the tube at a high speed, like 13,000 rpm for 10 minutes at 4°C. This will separate the mixture into three layers: an upper aqueous layer, a middle protein layer, and a lower organic layer. The metabolites are mainly in the upper aqueous layer. Carefully transfer the upper aqueous layer to a new tube. You can use a pipette for this, but be super careful not to disturb the other layers.
3. Sample Preparation for Analysis
Once you have your metabolite - containing aqueous layer, you need to prepare it for analysis. First, dry the sample using a vacuum concentrator. This will remove all the solvents and leave you with a dry residue of metabolites.
Reconstitute the dry residue in a small volume of a suitable solvent, usually a mixture of water and acetonitrile. The volume depends on how much sample you had originally and the sensitivity of your analytical instrument. Filter the reconstituted sample through a 0.22 - μm filter to remove any particulate matter. This is important because particles can clog your analytical column and mess up your results.
4. Analytical Techniques for Metabolomic Screening
There are a few different analytical techniques you can use for metabolomic screening of Tet - 213 cells. Two of the most common ones are mass spectrometry (MS) and nuclear magnetic resonance (NMR).
Mass Spectrometry (MS)
MS is a really powerful technique for metabolomic screening. It can detect and identify a wide range of metabolites based on their mass - to - charge ratio (m/z). You can couple MS with chromatography, like liquid chromatography (LC) or gas chromatography (GC), to separate the metabolites before they enter the mass spectrometer.
For LC - MS, you inject your prepared sample into an LC system. The LC column separates the metabolites based on their chemical properties. As the metabolites elute from the column, they enter the mass spectrometer. The mass spectrometer ionizes the metabolites and measures their m/z values. You can then use software to analyze the data and identify the metabolites.
Nuclear Magnetic Resonance (NMR)
NMR is another useful technique. It works by placing your sample in a strong magnetic field and applying radiofrequency pulses. The nuclei in the metabolites absorb and re - emit the radiofrequency energy, and the signals are detected and analyzed. NMR has the advantage of being non - destructive, which means you can reuse your sample if you need to. It can also provide information about the structure of the metabolites.
5. Data Analysis
Once you have your data from the analytical instrument, it's time to analyze it. There are a bunch of software tools available for metabolomic data analysis. Some popular ones are MetaboAnalyst, XCMS, and MZmine.
These tools can help you identify the metabolites in your sample by comparing the m/z values or NMR signals to a database of known metabolites. They can also perform statistical analysis to find differences in metabolite levels between different groups of samples. For example, if you're comparing Tet - 213 cells under normal conditions and cells treated with a certain drug, the software can tell you which metabolites are up - or down - regulated in the treated cells.
6. Validation of Results
After analyzing your data, you need to validate your results. One way to do this is by using standard reference materials. You can purchase pure standards of the metabolites you've identified and run them through your analytical system under the same conditions as your samples. This will confirm the identity of the metabolites.
Another way is to repeat the experiment. Do the whole metabolomic screening process again on a new set of Tet - 213 cells. If you get similar results, it means your initial findings are reliable.
7. Some Useful Peptides for Related Studies
During your metabolomic screening of Tet - 213 cells, you might also be interested in studying the effects of certain peptides. For example, Urocortin III (human) has been shown to have various physiological effects and could potentially interact with the metabolites in Tet - 213 cells. Another peptide is Substance P (7 - 11), which is involved in pain signaling and could also have an impact on the cell's metabolism. And Fibrinopeptide A (human) is related to blood coagulation and might play a role in the cell's microenvironment.
Conclusion and Procurement
Performing metabolomic screening on Tet - 213 cells can be a complex but rewarding process. It can give you a lot of valuable information about the cell's metabolism and how it responds to different stimuli. If you're interested in doing this kind of research and need high - quality Tet - 213 cells, I'm here to help. I'm a reliable supplier of Tet - 213 cells, and I can provide you with cells that are in great condition and well - characterized.
If you have any questions about the cells or the metabolomic screening process, or if you're ready to make a purchase, feel free to reach out. I'm happy to have a chat and discuss your specific needs.
References
- Wishart, D. S., et al. (2018). HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Research, 46(D1), D608 - D617.
- Dunn, W. B., et al. (2011). Guidelines and considerations for the use of system - level metabolic profiling in biomarker discovery and validation. Nature Protocols, 6(7), 1060 - 1083.
- Oresic, M., et al. (2008). Current trends in metabolomics for systems biology. Current Opinion in Biotechnology, 19(1), 17 - 22.