Camptothecin Apoptosis

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Uzair
Uzair's picture
Camptothecin Apoptosis

Hi I have rat periodontal cells growing in 24 well plates I think they will be confluent in a day or two and after that I have to induce apoptosis with camptothecin. I have never measured apoptosis in cells. If you could tell me how to go about with it, it would be great. I will be usung a concentration of 4 micromoles for 24 hours. I also want to know what I need to check under the microscope. I hope this is not a very silly question but I am basically new with research and I think you all are doing a good job and can help me. Thank you.

Raashmi
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Dear Uzair,

Dear Uzair,

No question is silly when it comes to science!!!
 If you are going to use Camptothecin then only 4 hrs incubation is sufficient to cause Apoptosis. To measure Apoptosis there are many kits available in market which are Flourescent based. If you have facility of FACS in your lab then it will be best to use Annexin V kit. Flow based analysis will give you best results to detect Apoptosis (Early as well as Late).

All the best.

Shubhangi
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Hi Uzair,

Hi Uzair,
Apoptosis is regarded as an early event so after 24 h incubation you may miss it. Drug concentration, cell number and incubation time all go hand in hand. Choose optimum of these three from literature. Or you can set up experiment and keep variable incubation time, say 4 hrs and 8 hrs.
Next is detection of apoptosis. It can not be done by simple microscope. Lot of kits are availbale that detect different apoptosis markers. one can use FACS as suggested by Raashmi. Homoghomogenous assay kits where no washing is requried are easy but expensitve .
Steps are :  add cells+ drug ---- incubate --- add kit substrate --- incubate or no incubation --- read luminescence or fluorescence
Hope you find it useful
Shubhangi

samm
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Hi Uzair, you can actually

Hi Uzair, you can actually see apoptotic blebs under the microscope when the cells are stained with a Hoechst or DAPI or AO DNA dye. However, the process is NOT very quantitative, and requires a trained eye, ideally 'blinded' to experimental conditions for scoring.
If you want to try a kinetic apoptosis assay, you can try the fluorescent plate based assays to measure active Casp3 in live cells such as FLICA - a series of wells in the same plate, with the same batch of cells and drug can then be used for the analysis.
I'd recommend trying out the FACS separately first - many adherent cell types expose significant amounts of AnnV on detachment - usually in a reversible manner (AnnV+/DNA frag-/perm-). Remember, you CANNOT fix these cells, so they have to be acquired within minutes of staining (you may have to stagger samples).
Finally, LDH release in sups+ active Casp3 in cells can be done as a dual assay on the same cells.

Uzair
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Thanks to all the replies. I

Thanks to all the replies. I really appreciate it.