Hi Anoop,
I would say it will depend on how critical it is to pinpoint apoptotic cell death (versus other types of programmed cell death), on the cell type you are studying and on what type of staining you used in flow cytometry. As a general principle I agree with the previous comments that it is always best to confirm your findings from one type of assay with a complimentary type of assay. As flow cytometry for cell death is concerned, keep in mind that propidium iodide (PI) staining alone can not distinguish between apoptotic and necrotic cells, and as cells in dishes often undergo secondary necrosis you will frequently see apoptotic cells stain with PI at later stages too. I would recommend you use double staining by annexin-V-FITC coupled with PI for flow, as that would indicate caspase activation. Maybe useful to further confirm caspase cleavage / activity by western blot or activity assay; using a pan-caspase inhibitor such as z-VAD. DNA laddering can be very helpful especially if you are studying insect cells, and it is quite inexpensive and fairly easy to perform. You may also want to check Galluzzi et al., CDD, 2009, for general guidelines on cell death assay use and interpretation. Good luck,
if your experiments focus on apoptosis, I think you need to use different kinds of techniques to explain it (at least two). for example, you can use confocal imaging to show the change of the cell nucleus.
Agree with Shuaipeng. Otherwise, flow cytometry is already quite conclusive.
Usually these type of experiments needs to be confirmed by at least two techniques. However, I think flow cytometry is good.
Hi Anoop,
I would say it will depend on how critical it is to pinpoint apoptotic cell death (versus other types of programmed cell death), on the cell type you are studying and on what type of staining you used in flow cytometry. As a general principle I agree with the previous comments that it is always best to confirm your findings from one type of assay with a complimentary type of assay. As flow cytometry for cell death is concerned, keep in mind that propidium iodide (PI) staining alone can not distinguish between apoptotic and necrotic cells, and as cells in dishes often undergo secondary necrosis you will frequently see apoptotic cells stain with PI at later stages too. I would recommend you use double staining by annexin-V-FITC coupled with PI for flow, as that would indicate caspase activation. Maybe useful to further confirm caspase cleavage / activity by western blot or activity assay; using a pan-caspase inhibitor such as z-VAD. DNA laddering can be very helpful especially if you are studying insect cells, and it is quite inexpensive and fairly easy to perform. You may also want to check Galluzzi et al., CDD, 2009, for general guidelines on cell death assay use and interpretation. Good luck,
Hi,
Yes. Showing the apoptotic laddering would be confirmatory and also give an idea of relative levels of fragmentation pattern of the DNA strands due to apoptosis in your experiment..
In fact, the response is "definitively yes" to your question.
To be convinced by my response, you should have an "in-depth" reading of the attached articles ...
Best regards
Robert
- Badges
- Science method