confirming how the gene causes decreased growth/increased cell death - (Mar/13/2008 )
What experiments can help me check how does a gene/protein contribute to either INCREASED cell growth or DECREASED cell death?
I see decreased growth on performing growth curve expts on my gene-specific siRNA treated cells compared to GFP siRNA treated cells (neg control),
indicating the mechanism is either gene causes cells to grow faster or die slower.
I hope I am putting across my question effectively? if any suggestions please reply.
You can do BrdU assays to measure progress through S phase (increased growth)
And/or apoptosis assays to determine if you are getting increased death
Cell growth assays you could use would be BrdU or MTT. Cell cycle analysis by staining with PI could also help, but won't tell you how fast cells are progressing through the cell cycle.
Apoptosis assays include annexin-V, TUNEL, sub-G1, caspase activation. There are also a lot of caspase independent cell death mechanisms you could look at like autophagy and necrosis. There are many possibilities regarding cell growth and death, so there is no all encompassing single experiment.
For cell growth rate, you can simply count the cell for certain time (e.g. every day for 3-5 days), or more accurately, perform 3H-thymidine incorporation assay.
For cell death, many different types of assay for apoptosis detection can also be used.
Thanks everyone for the suggestions... i shall try working on those..firstly getting to understand their protocols!