I am going to use a chemical to induce apoptosis. I am wondering how I could know if my apoptosis is induced successfully.
Hi,
Depends what you are more familiar with.
If you like FACS, then go for AnnexinV/PI staining.
If you like microscopy, then use Hoechest staining and look for Nuclear condensation. You may observe crescent shaped nuclei, And occasionally you may see some fragmented nuclea. for fragmented nuclei you will have to do time wise treatment. Because more fragmented nuclei may be lost at latter time points of apoptosis (Sec Apoptosis).
If you like Western, Then either go for Caspase3/8 or PARP cleavage. Once again you will have to chose at least two time points. Because activated caspases are transiently observed and some times Cleaved fragment of PARP is also further degraded.
Good Luck
You can stain your cells with 5 ug/ml Hoechst 33342 added directly to the medium (without its removal). Incubate it 30min in the incubator. Then observe under fluorescent microscope whether or not there are visible apoptotic bodies. The second method is measurment of caspase 3/7 activity. Good luck!
Hi,
Depends what you are more familiar with.
If you like FACS, then go for AnnexinV/PI staining.
If you like microscopy, then use Hoechest staining and look for Nuclear condensation. You may observe crescent shaped nuclei, And occasionally you may see some fragmented nuclea. for fragmented nuclei you will have to do time wise treatment. Because more fragmented nuclei may be lost at latter time points of apoptosis (Sec Apoptosis).
If you like Western, Then either go for Caspase3/8 or PARP cleavage. Once again you will have to chose at least two time points. Because activated caspases are transiently observed and some times Cleaved fragment of PARP is also further degraded.
Good Luck
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