Transfection - (Sep/12/2009 )
I am doing a luciferase on 293 cells. I am told not to transfect with too much DNA or cells will die (and not to stimulate with too much TNF-a for same reason. My question is why do we care if cells die as we are finally going to lyse and kill cells anyway. We incubate for 48 hours with our plasmids. As long as cells live sufficiently long for plasmid to get in and express luciferase, we should not care - right? Why are we so concerned for cell death?
because when you harvest the cells you will remove the dead cells - so they won't figure in the assay, this artificially alters the results of the experiment unless all the cells (including controls) are treated exactly the same. The cells need to be alive (as in alive when lysed) for the luciferase to work - dead cells break down proteins when they are dying.
but if dead cells denature their proteins while dyeing....then even lysed cells should have their protein denatured, right? I can understand that harvesting (or washing he 24 well plate with PBS) will wash away the dead cells. But if I do not wash the medium with PBS, and directly add the Passive Lysis Buffer into the cells (in media) before testing them for luciferase, then it seems to me that dead cells should not matter. But obviously dead cells do matter. So I want to know the reason why?
I used luciferase reporter system from promega, and PBS wash before adding lysis buffer definitely affect my result. Do you follow manufacturer's direction to add lysis buffer directly to culture media? I got better result when I wash cells with PBS (once or twice), pour lysis buffer, store -80C, thaw and spin before assay.
Lysing cells is quite different from the cell dying by apoptosis or necrosis - in both of the latter cases the death of the cell causes the proteins to be broken down (degraded, NOT denatured). When you lyse the cells the lysis breaks down the membrane and dilutes out the proteins and you will usually add protease inhibitors to stop the proteins being degraded. At this point the proteins can be either retained in a native configuration (this is what you will have for luciferase), or denatured (e.g. for western blotting) depending on the lysis buffer you have added. The important point is that the proteins from lysed cells are intact and ones from dead cells are usually not.
So in your experiment you will have controls right? If you don't you are definitely doing it wrong! The controls should be: Untreated cells (no plasmid, no transfection reagent), Transfection reagent only, and Empty vector (luciferase plasmid with no gene of interest). So if you transfect your cells and the level of DNA you use causes 50% of the cells to die in the treatements (and remember you won't know this proportion of death unless you count your cells)- how will you compare that to the controls where less than 50% will have died?
I add lysis buffer directly to culture media. But I think that washing it with PBS should give better results. So what do you do? You wash with PBS before adding lysis buffer. In a 24 well plate, how much lysis buffer do you add (I add 50ul)? After that, do you measure luciferase? Or do you freeze with your lysis buffer (in the wells along with the cells), then thaw it (thereby lysing it even more), and finally take luciferase?
Thanks, this was very helpfu. If I understand it right, what you are saying is apoptosis or necrosis degrades the protein, while lysis (if done correctly) does not degrade the protein. This makes total sense. However, why are proteins degraded on apoptosis/necrosis, once the cell dies - the protease (which is a protein as well) should be degraded and thus not active, right? Hence, it should not be able to degrade other proteins.
Another qs: I understand that apoptosis might lead to degradation. So is it correct, that if the cell dies immediately on adding my DNA, then the proteins will surely be degraded (as I harvest after 48 hrs). However if the cells are apoptotic only at the end (say after 48 hrs), then apoptosis should not matter so much?
I think the real issue is that you need to have cells that survive the transfection, and those tranfected cells should presumably represent the biology you are studying accurately (which seems to be TNF induction, not other mechanisms of apoptosis). While the luciferase assay is extremely sensitive, it should be possible to tranfect your DNA without major toxicity (especially in 293 cells) to yield a robust culture of transfected cells.
Also, it seems that you are using a two step luciferase assay (where a lysis buffer is added and a sample of lysate is used for teh luciferase assay). With this type of assay, it is crucial to wash the cells with PBS before adding the lysis buffer. There are culture components that will effect the luciferase assay in the media. In the add and read assays such as Steady-Glo or Bright-Glo these components can be dealt with due to the chemistry invovled. That being said, phenol red absorbs a significant portion of the luciferase signal in either case and should be removed in either case.
Hope this helps.