We observed some dose dependent cell death in Hela cells after treatment with a potential drug we developed. We want to know what kind of cell death it induces, but without having to dive right into elaborate assays such as FACS (as we need to screen multiple cancer lines to confirm). Thank you!
-- Please suggest some assays (pros and cons): e.g. TUNNEL, WB for caspases, DNA fragmentation, some commercial kits (esp. if you have done or used!)
PI-FACS is the simplest assay to test apoptosis vs necrosis. Then it would be appropriate to go for further assays.
jinesh
First you also can check by Sytox stain. And after that I would go more in detail by P/I.
Annexin V-FITC is the most simple test...the membrane defect is the first to happen before everything else! Please review your cell biology 001...floppase and flippase, etc
Acridine orange-ethidium treatment as stains are interesting but tehy may have a toxic activity on DNA through groove insertion...the annexin V test is more mild to test injurious actions on treated cells witout affecting change in nucleus.
Thank you, Jacques, for the explanations. We will consider exploring annexin V as an assay.
Apoptosis: You could start off with DAPI staining to look at nuclear fragmentation. WB for PARP cleavage and caspase 3,8,9 will give you an idea of caspase dependent mechanisms. Another easy assay for cell death/apoptosis is sub-G1 analysis with PI staining.
If you observe apoptosis but no caspase activation you can look into caspase independent apoptosis mechanisms.
To rule out necrosis/necroptosis the best assay would then be annexin V/PI staining
Excellent comments, thank you Varun. We can look into those when detailed mechanistic study is called for.
Apoptotic process is a "process". Cell-to-cell variations exist when a cell population is treated with an apoptotic inducer, eg cell fate (to die or to survive); time delay between exposure to inducer and the activation of effector caspase / mitochondrial outer membrane permeabilization; to die of apoptosis or other cell death mechanisms. Since the cells progress to apoptosis heterogeneously, two or more assays are usually recommended to confirm apoptosis. Definition of cell death subroutines are always under revision and updated. And people have found that some apoptotic markers, eg Annexin V/PI, caspase 3/7 activity, can also occur in other cell death subroutines. That's also why you should perform at least 2 assays. You can refer to this review in the link for nomenclature of cell death.
Go back to your question. I agree with Varun to start with DAPI/Hoechst staining to look at nuclear fragmentation/condensation. But before that, I would first observe the cells under phase contrast light microscope for any cell shrinkage/rounding, membrane blebbing, and even apoptotic bodies formation. If your cells do show these features, go for nucleus staining, and then WB of PARP and caspases, Annexin V/PI, caspase activity assay, PI staining for cell cycle analysis, etc.
http://www.nature.com/cdd/journal/v19/n1/full/cdd201196a.html
The appropriate biomarkers include Annexin V for early stages and Caspase 3/8,, BCL-2 and Fas the tunneling assay for late stage events. Why Hela cells and not some other tumor cell line? Hela is infected with HPV16 and is extremely high passage (since 1950's). I would check the literature for additional cell lines for comparison. Alsom the Akt?MTor pathways may be involved.
Thank you Bob for pointing out the problem. Good suggestions. We plan to validate the compound's effects in more "refined" cell models other than Hela.
Have a look (or maybe a "profound" reading) of the attached articles.
Best regards
Robert
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