Hi all,
I'm currently an undergraduate working with EBV transformed B cells (GM12878). I am attempting to induce autophagy and LC3 positive autophagosome accumulation through starvation and chloroquine treatment. Cells are then stained with a FITC conjugated anti-LC3 antibody as dictated by the protocol of the FlowCellet LC3 Detection Kit.
When imaging using a confocal microscope there is no clear LC3 signal, and definitely no LC3 positive punctae as you would expect to see. I have tried extending the starvation/chloroquine treatments across of a range of 3-8 hours with no change in results.
Does anybody have any ideas as to why I cannot get a clear LC3 signal?
(I should also add that a second round of staining against a different target is performed after fixation.)
The cells were subsequently fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100?
When you would like to observe LC3 puncta formation, you have to use Saponin or Digitonin instead of 0.1% Triton X-100.
Hi, thanks for your response!
So prior to LC3 antibody staining, a proprietary selective permeabilization buffer from the kit is used. It's not stated what the content is, but from the description of it's action it appears to act in the same way as saponin (allowing LC3-I to be washed out of the cell but preserving LC3-II).
After this I have been fixing the cells as you described to prepare for a second round of staining against another intracellular target. The permeabilization buffer for this round of staining is 0.1% Triton.
Is this a problem? I didn't think it should disrupt the LC3 stain as the cells have already been fixed at this point?
To summarise how I've been working on this (and there will probably be something stupid here I have overlooked):
1. Autophagy induction (Starvation w/ chloroquine)
2. Selective permeabilisation (Likely a saponin based agent)
3. FcX block and primary LC3-II staining
4. Fixation
5. Permeabilisation (Triton X-100)
6. FcX block and primary stain of 2nd target
7. Secondary stain
8. Mount and image.
Triton X-100 is considered to be the cause of the failure!
Triton X-100 destroys LC-3 puncta.
Hi again!
I shall run an experiment tomorrow replacing the Triton with what I believe to be a saponin based agent. I have had a quick look through the literature and cannot find any specific reference to a destructive effect of Triton on LC3 punctae.
Would you be able to supply any publications that support this? I've seen several publications where Triton is used for LC3 puncta detection seemingly without issue?
Dear Charlie, I have used 0.25% Triton X-100 for over 8 years and always got reproducible results using Chloroquine (CQ) on Jurkat T-cells and myleoid cell line K562. I would suggest maybe it is the 4% PFA you are using is to high for suspension cells I have used 1% PFA for the cells mentioned above and using 4% PFA on suspension cells makes them very small not ideal for imaging. But the 2 cell lines I have used respond very differently to CQ using 25, 50, 75 & 100 uM and fresh blood monocyte respond maximally at 10 uM CQ (for 24h) so I wondered if you had tested a range of [CQ] you may find a maximal response at a very different CQ concentration. Also FITC is not the best fluorochrome for imaging could you switch to AF488 or 647 for example
REgards
Gary, Blizard Institute, Queen Mary University London
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