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I work with 3T3-L1 cells, but in 12- or 6 well plates. At completion of my experiment, with culturing and treatment, should I harvest the cells, spin down, remove the supernatant and then add the 100 µL Lipid Extraction Buffer? Following which I heat and mix as in 1a iv. and v. (page 7), and then centrifuge to remove debris?
Although I have not counted, I am pretty sure a 6-well plate will give me more than 10.000 cells per well. If I plan to perform the triglyceride assay, should I then transfer 5 µL of my lipid extracts (from cells grown in 6- or 12-well plates) to a 96-well plate, or would you think this still exceeds the standard curve?
Asked on Jul 17 2013
In our hands, ˜1,000-10,000 differentiated 3T3 cells using 100ul Lipid Extraction Solution are sufficient for the colorimetric assay. Then we use 5-50ul of the extract for the assay. I would start with 5ul of the lipid extract for the assay. If this is too much, then I would do a pilot experiment using several dilutions to see which one gives me the best results within the linear range of the std. curve.
The protocol you mention is correct:
After culturing and treating your cells in a 6 or 12 well plate, you should remove the medium, wash with PBS, add 100ul lipid extraction buffer, heat for 30 mins, cool to RT and then use the liquid for assay. You can spin down to remove any debris.
Elisa ThomasAbcam Scientific Support
Answered on Jul 17 2013