I am transiently transfecting HEK293T cells with pEGFPC1 WT TDP-43 construct using PEI transfection reagent. After 40hrs of transfection, I am staining the cells with annexin v apc and Propidium iodide and measuring the fluorescence by Flow Cytometry. But the annexin v and Propidium iodide binding is not significantly increased as compared to empty vector transfected cells. What parameters should I change to get a significant annexin v and Propidium iodide binding in TDP-43 transfected cells??
Hello, Madhuparna!
You do not get an increase in PI and annexin staining of cells transfected with TDP43, since this protein is a DNA double break repair protein, and annexin and PI are dyes for apoptosis.
https://en.wikipedia.org/wiki/TAR_DNA-binding_protein_43
Thus, TDP43 restores DNA breaks, that is, inhibits the initiation of cell apoptosis, so you don't see aopoptotic cells when stained with PI and annexin.
Then what technique should I use to determine TDP-43 over expression mediated cell toxicity in HEK293T cells?? @ Alexandr Chernov Sir
If I understand correctly, your assumption is that transfection of TDP-43 would cause apoptosis but this protein is an RNA-binding transcriptional regulator with no obvious triggering of apoptosis, except in some protein aggregation neuronal degeneration diseases. Please read the paper where they observe TDP-43 aggregation and neuronal apoptosis with increased levels of zinc. So adding zinc to your cells might be worth trying.
Another comment is related to the use of PI which enters cells with broken membranes but apoptosis is not characterized by leaky membranes in contrast to necrotic or pyroptotic cell death. So PI might not be a good early marker of apoptosis. There are a couple of reagents that you could use to detect activation of the executioner Caspase 3 (please see references).
Do you see good fluorescence with EGFP? EGPF cloned in the N terminus hopefully does not interfere with dimerization of TDP-43.
Doesn't the PEI reagent have background fluorescence?
Can you use some reagent like TNFa, CD95L or anything that triggers apoptosis in your cells? That way, you'd have a positive control for changes in Annexin V and PI related to apoptosis. Does it increase with EGFP-TDP-43?
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Article Zinc induces depletion and aggregation of endogenous TDP-43
https://www.nature.com/articles/ncomms3157https://www.merckmillipore.com/BE/fr/product/Caspase-3-Detection-Kit-Red-DEVD-FMK,EMD_BIO-QIA93?ReferrerURL=https%3A%2F%2Fwww.google.com%2F
I have used doxorubicin treated cells as positive control. They gave a lot more Annexin v and PI binding as compared to TDP-43 transfected cells.
I have also tried with pcDNA3.1+ FLAG TDP-43 construct but the result is same.
@Oscar L. Sierra Sir
Madhuparna Chakraborty , hi, great! you have an excellent positive control that triggers apoptosis that can be detected by the readouts you're using.
Now, you can take some transfected cells with EGFP alone vs EGFP-TDP-43 and treat them with doxorubicin. In the EGFP alone, Doxorubicin should trigger apoptosis just as you're currently observing. But it's possible that EGFP-TDP-43 cells undergo even higher apoptosis rates based on your assumption that this gene should increase apoptosis. Make sure to compensate correctly in order to avoid the EGFP bleeding into the PI (red) channel.
For Annexin V, I'd do the same but without any EGFP, just regular plasmids. Control plasmid could be pcDNA3.0 or any other you have; test plasmid could be CMV-TDP-43. Treat with Doxorubicin and evaluate by Flow Cytometry in the FITC channel.
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