I'm knocking down a gene in different cell lines and 24 h after transfection all that I can see is cell debris, vesicles (which I assumed that were apoptotic bodies), and some intact single cells here and there in treated wells.based on these observation I assumed that my manipulation caused cell death. however, PI cell cycle analysis did not show any significant accumulation in sub G1 phase (only 7% increase in sub-G1 cells) or even cell cycle arrest in other phases. I also tried out viable staining using trypan blue but apoptosis rate was not that much increased in KD wells, although number of cells in KD wells was reduced drastically compared to mock transfected wells. any idea what is going on here? should I collect my cells sooner than 24 h post transfection to see the increased apoptosis or any other changes?
hi
1- optimize best concentration of chemical reagent for cell transfection ( lower toxicity with higher rate of transfection such as polymeric based trasfection reagent )
2- check transfection rate by GFP vector
3- check 4 . 8 , 12 ,24 hour after transfection
4- check cell apoptosis ratio by AnexinV-FITC kit
PI dye only positive in late apoptotic and necrotic cells but negative in early apoptotic cells .
good luck
Thanks for your tips, well, I've already optimized the concentration of the transfection reagent and it works fine in my mock wells (shows no changes in cells morphology and viability). I'm gonna try anexinV kit as soon as I can get my hands on,but I was wondering if I could get a possible explanation using viability/proliferation tests.
Even if the degraded DNA in Sub-G1 is not as strongly increased as expected after 24h you might have apoptosis. I have seen it before in my hands, cells need to be strongly damaged and in advanced stages of apoptosis in order to detect DNA fragmentation by PI-FACS. You should still check by Western of markers such as cleaved Parp1 or cleaved caspases 3. However, i don't know your detailed experimental settings or aims, but you might also be looking at other cell death programs e.g. autophagic programs, here a review: http://jcs.biologists.org/content/127/10/2135.
Good luck with your research!
You can also use Hoechst 333424 for a life-time detection of apoptosis and you can try to analyze after 48 h. If apoptosis occurs, you will discover chromatin condensation and nuclear fragmentation. Usually, this is observed together with an increase in the sub-G1 phase.
Good luck!
Dear Scientist,
in respect to your questions there are many possibilities why you lose your cells after transfection. As previously mentioned:
1. the conditions for transfection is important, but this you have checked (mock control didn`t show this lost of cell viability!?)
2. Your transfected vector with the gene of interest seems to induce the morphological changes. Here I would check the characteristics of the morphological and biochemical changes in a kinetic manner (24h is to late). If you see membrane blebbing then the cell undergo apoptotic cell death. If not then non-apoptotic cell death occure: e.g. ferroptosis, patanatos, necroptosis (also programmed but from a morphological characteristic equal to necrosis) and others. Check the paper from Galluzzi et al.,. Many experts work in cell death field discribe in this paper (review) biochemical and morphological characterisitcs of the different cell death types.
3. You can also use inhibitors of caspases (zVAD, QVD) or RIPKinases (e.g. Necrostatin-1) to investigate if you can safe your cells from apoptotic or necroptotic cell death. Here you can get indirect and additional information about the possible cell death.
4. You want study the function of your gene of interest. Here a constitutive expression vector model is not suitable to identify the functional relevance of your gene of interest. Try to use an inducible gene expression vector system, if your gene of interest induce cell death or senscence or cell cycle arrest in different manner. This would allow you to study the function of gene of interest in better manner.
All the best and much success for you!
- Badges
- Science method