I'm setting up AO staining to detect autophagy. The first observation, Tamoxifen-induced autophagy on MCF-7 cells had many huge cells (figure attached). Is that normal in autophagic cells or not?
First of all, good luck with science.
I dont think they are 'cells' inside cells. they can be autophagic vacuoles, but you should compare with the living ones.
In case you find cells inside other cells, it can be entosis!!! but here I dont think you can observe it clearly only with AO staining.
You can check my publications in order to have more detailed information autophagy in cells (including MCF-7 cells) and how to observe autophagy with different stainings e.g. anti LC3 antibody based, AO.
Best Regards,
Dr. Gizem AYNA
All that I can come up with off the top of my head is that tamoxifen has cytostatic effects (rather than cytotoxic) decreasing divisions of the cells which might possibly lead to increased cell size in the sample.
I agree with Mar Eglisson...large cells could be cells in G2/M phase of cell cycle....to confirm this hypothesis you can add nocodazole (0.4 μg/ml) to MCF-7. Hello and good luck.
It seems that you may care about the position of your cells within the cell cycle.
Always compare your treated sample with control untreated cells.
I you have problems, go to cell synchronization and repeat the experiment.
Look carefully to your picture... Your cells are mostly homogeneous in size... Except the biggest one which is certainly in G2/M phase as ascertain by Maria Russo.
Be careful with AO staining. and also go for more safe experiments... EM picture might be helpful as well as LC3B-GFP expression and other .... experiments. There is a nice paper in autophagy... summarizing all the need for autophagy detection and analysis built up by Klionsky's and associates.
Thanks for all. I really appriciate that. I'm going to learn more...
I compared the Tamoxifen-treated MCF7 with untreated MCF-7. I only saw the huge cells on treated cells. Beside that, when I observed the huge cells, I saw red-flouresence dots inside the cells (but in my picture, you can not see that becasuse my microscope just paint one color - AXIO Scope A1).
Can they have autophagy when they in G2/M?
Autophagic vacules stain red/ orange by AO staining. AO stains acidic compatments as red fluorescing structures. Since there is no autophagosome specific dye so AO and MDC are generally used. The pic u have uploaded does not indicate autophagy. it merely shows some vacuoles with neutral pH. you can check the number of these giant cells if it is 2-3 % i think it is not a problem to worry about.
you can validate ur results using bafilomycin A1 and CQ by AO staining and LC3 by Western. use Wortmanin or 3-MA as negative controls. Also u can check whether starvation or Rapamycin also increases the no of giant cells. if These two known autophagy inducers have no effect on cell size then it is a good finding, the effect may be specific with ur drug....
Good luck dear
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