We want to standard curve for luciferase assay by using a range of 5x106 to 1 cells / well in 96 well plate. We will use Kit from Promega.
My question is since upper limit is too high to high to seed cell overnight for adherence, then how can we remove media as the protocol says to remove media and wash cells?
In case if we do not remove will that be OK?
last question is if not to remove media is OK, then do we use the same ratio of reagents e.g. according to the protocol lyse the cells in 20ul and after the cells are lysed add 100ul of luciferae reagent.
I'm not entirely clear what you are trying to achieve. Some more details would be helpful. Eg. are you transfecting in a reporter? Are you doing a dual luciferase assay? Why do you need so many cells that they are too dense to seed overnight?
Generally, the linearity of luciferase assays is so good that many people don't use a standard curve.
With regards to your question about whether the media has to be removed. With a standard luciferase assay I'm pretty certain it wouldn't work very well if you didn't remove the media. For a start, your cell lysate would be very dilute due to the presence of the media.
I know that Promega make a dual luciferase assay kit that you can be used directly on cells in medium without prior lysing. I don't particularly want to advertise one supplier but you mention you're using Promega. Their homogenous assay is called Dual-Glo, although I'm sure other companies will make this sort of assay too. These assays may be more suitable for you.
Non adhered cells would behave physiologically different, so your highest density well would be your curve limit. If you are truly wanting a reading from a higher cell number per well then you could possibly add an extra step in the protocol wherein you aspirate the media and centrifuge the media and non-adhered cells. Add the lysis buffer to the cell pellet and pipette the lysis mixture from each tube back into corresponding well of your plate and continue with kit protocol. Again, non-adhered cells will behave differently than cells in a mono-layer. Of course you could possibly utilize non-adhered controls to normalize.
"@ Timothy J Pullen Thanks a lot for explaining and asking me for detail.
Actually, we already have developed stable cell line for Luc, in next step we want to implant a xenograft of these cells in mouse model. Although it would not be good enough, we want o get an idea how many cells we can detect in-vitro to project similar results for in-vivo study.
We already had planned to go for lysis then making serial dilution to get hypothetical range of 1x106 to 1 cell per well so media would not be a problem.
We will use dual luciferase kit from promega but will be looking for single luc system.
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