hi to everyone,

i want to discuss ne of my prob in the hope that i will get something very imp clue for my recent prob.

actually i am seeing the antitumr activity of one herb on one tumor cell line. first i did the MTT asssay. and by this i found that this drug is inhibiting the proliferation now i wanted to seee the dna fragmentation:rolleyes: unfortuantely at all conc only iget the one band of genomic dna, and no fragmentation.

now i want to ask that

1.whether it is possible that there is apoptosis but no dna fragmentation.
2. will dna fragmentation occur in both intrinsic as well as extrinsic pathway.
3. why we use the DMSO for the treatment of drug extraact, is there any other replacement for it.
4. how the apoptosis cell will appear in the inverted microscope.
5. will the isolation of the genomic dna and the fragmented dna will be same or different, i use the same protocol.
6 and when we are preparing the drug( having polyphenol) then what shouid be our strategy and what precaution we should take.

all the comment are most welcome and thanx in advancer for the act of cooperation.

I feel that checking for DNA fragmentation is often more troublesome then other commonly used methods to check for apoptosis. If you were specifically interested in nuclease activity I'd recommend the TUNEL assay instead, as its more sensitive. DNA cleavage is a common defining feature of apoptosis, though cell death can be occuring without apoptosis or DNA damage. An apoptotic cell will be shrunken with marginalized chromatin (as viewed with DAPI or Hoechst dyes), and will over time form and release apoptotic bodies.

Videos and images of what I just described can be found here.

As far as whether DMSO is necessary - without any info about the chemical you are testing we would have no idea about other alternative solvents. Small amounts of DMSO in tissue culture media (<0.8%) are relatively non-toxic to cells. Just perform the correct controls and you'll have no worries.

Protocols for DNA extraction can be found in Maniatis, et al. or on google.com. I hope this helps