Hi,
I am starting studying Mitophagy, so I was looking into some publication to see how to measure mitophagy reliably. Like which kind of markers do you usually use?
is colocalization of LC3B and TOMM20 a good strategy?
Hello Federica Albanese
Please refer to the research article given below for more information on Mitophagy.
Article Tools and techniques to measure mitophagy using fluorescence...
Best Wishes.
Colocalization of autophagy markers and any mt markers, e.g. vdac, is normal, but requires some controls (autophagy blockers vs autophay inducers). TEM imaging can give you a rough estimate of the number of mitophagic events per single cell slice. need to account fission/fusion rate, which is cell dependent/condition dependent
Thank you so much both for your help! My plan was to mark both LC3 and Tom20 and Lamp1 and Tom20 and treat cells with FCCP (mitophagy inducer) and CQ (autophagy blocker) , in order to see how many mitocondria are engulfed in autophagosomes and how many mitocomdria are effectively delivered to lysosomes. Reagarding this, I have a question: do I need to transfect cells with GFP-LC3 or detection of endogenous LC3 would work either way?
Plus is tom 20 a bad marker, should I pick VCA1 instead? Also to determine mitocondria fission and fusion events, is TEM the only tec ttecnique I can use?
Should work with any "housekeeping" mt protein, I guess. I did not use Tom20 or other TOMs 'coz I did not have these Abs at hand + VDAC was cheaper.
for some reason, endogenous LC3 gives weaker signals on IF, than when expressed any flag-plasmid. In addition, GFP-LC3 will help to test autophagic flux which many reviewers should ask you too.
Hope it helps a bit:)
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