Triglycerides in culture medium - (Jul/18/2006 )
We're going to measure the total triglycedide content in culture medium and/or cell by using the Serum triglyceride determination kit (TR0100 Sigma Aldrich).
My cells are adipocytes 3T3L1.
Mya, not sure what your question is really but this may help.
You can measure TRIGS by chemical or enzymatic methods. Chemical methods need a solvent extraction step to solubalize the TRIGS and denature interfering proteins. You release glycerol from the TRIGS by saponification (use KOH). The glycerol is then oxidised to formaldehyde with Na-periodate. Formaldehyde then reacts with acetylacetone and ammonium to form a fluorophore. I think you can also react the formaldehyde with chromotropic-sulfuric acid which then absorbs at 570nm.
Enzymatically, lipase breaks down TRIGS to yield glycerol. Glycerol then reacts with ATP to form ADP and glycerophosphate. The ADP is reacted with phosphoenolpyruvate with pyr kinase to give back ATP & pyrvate. Finally the pyr reacts with NADH to yield lactate & NAD. You measure the disappearance of NADH at 340nm.
If you are using a serum kit, just check its validity against the medium you have suspeneded your cells in. Should be ok but worth checking and doing a neg control.
Let me know if you need details for the assays.
The question really was: how to prepare the sample to proceed with the assay?
This kit has been set up for triglycerides in seum and blood. We're going to use it for triglycerides in medium and cells