I dump certain steroid treatments on my cancer cell lines followed by annexin staining. However even if i am observing under the microscope that there is significant cell death as all cells have clumped up and are floating freely but in annexin staining i am not picking up significant stain.
Why is it so?
Like measures of cell viability, no single parameter fully defines cell death in all systems; therefore, it is recommended to use several different approaches when studying apoptosis. Is it possible that your steroid treatments are inducing necrosis and not apoptosis? If not, then is the timing of your staining appropriate?
Try Trypan blue, if they stain, its confirmed Necrosis. Annexin stains phosphoserine in Apoptosis. Also, re-suspend them in DMEM or RPMI. Steroids can cuase excitotoxicity.
Hope this helps.
Best,
Mannan
Annexin staining is indicative of apoptosis; it sounds like the cell death you observed is of some other type, possibly necrosis.
You can try congo red or trypan blue (filter solutions after preparing them). Hope it works for you! Irene
You should try a double staining; for instance Anexin V and propidium iodide (PI). PI will label all died cells, either necrotic or apoptotic, whereas annexing V will label only apoptotic cells. So, Anexin V+PI+ double positive cells are bona fide apoptotic cells; Anexin V+PI- cells are cells committed to apoptosis. You should do a kinetics analyzing cells at different time points after stimulus addition.
Good luck!!
Dear Moumita,
As previously other colleagues indicated there are different types of cell death. Annexing V will label only apoptotic cells. Trypan Blue or Congo red stain confirmed Necrosis but I think it is needed a kinetics analyzing cells at different time points after stimulus addition. By other hand anothers types of cell death are possible.
Good luck, Joan.
Dear Moumita,
As suggested by many colleagues, you should verify the results using other methods of apoptosis.
But, based on your observation of clumping, I would suggest apoptosis will always not result in clumping. Clumping during typical cell culture exp may also due to (i) change in surface charge of cells due to treatment, (ii) massive/quick action of some cytotoxic agent, and (iii) a basic property of certain cells.
Non-annexin staining of cells will be may be due to very advanced stage of cell death, where due to membrane debris, you may not able to visualize the annexin staining. Or cell death is following necrosis path, where phosphatidyle serine externalization is very minimal.
So I would suggest first check by trypan blue, whether there is necrosis, second check from literature about the properties of cells/nature of steroid you are using.
Hope you will solve the issue with systematic ruling out the possibilities.
Best wishes,
Badri
Thank you all for the valuable suggestions.
As mentioned by most even i can figure out that i am not picking up annexin staining may be because of the advanced stage of death of my cells where due to excessive membrane damage may be the PS and annexin binding is failing to happen.
I have planned to reduce my treatment time and see what happens.
Its a very preliminary experiment for me and hence i didn't go for dual staining because my objective was to check toxicity of my treatment at first place.
Also can someone forward me some articles which states low annexin staining in late stage necrosis. I am looking for some but i haven't found anything impressive.
Thanks
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