Has anyone used a lysotracker to follow lysosomes staining during autophagy? And also follow that by image and flow cytometry analysis?
Any suggestions are welcome but information related to real experiments will be appreciated. The idea is that the staining will increase until a point where the lysosomes associated to the autophagosome and the staining might decrease.
Dear Patrice,
We are currently performing exactly the same experiments you are planning to do ! We use Lysotracker Red to visualize lysosomes in drug treated living cells and analyze cells by microscopy. According to Invitrogen Lysotracker is not suitable for flow cytometry. To confirm our data we are staining fixed cells with a LAMP1 antibody to compare the results of living cells with fixed cells. We are doing this because Lysotracker generally stains all acid organelles. We are staining autophagomes using the classical LC3 staining respectively western blotting. If you have a specific question, please ask!
Kind regards,
Robert
Dear Dr Petit,
we have worked with Lysotracker Red in plant root tissues to visualize autophagosomes, although as already stated LR stains pretty much all acidic compartments. we also used a cysteine protease inhibitor to retain autophagosomes; otherwise we could not really detect much. I don't know if it is applicable or related to your situation, but I wanted to share.
good luck with the experiments,
Dear Patrice,
yes, care needs to be taken when using lysotracker only, to assess autophagic activity. I suggest a combination of imaging an flow cytometry. the lysotracker signal is not always as responsive, and is only an indirect indication for autophagoic activity. We usually do GFP-LC3 transfects, LC3 blots, and importantly, with and without bafilomycin, to assess the change in autophagosomal degradation/synthesis. the tandem constructs are also nice, but need good verification with colocalization analysis. We have also used the lysotracker sucessfully on the flow, but then also in addition to LAMP asessment. If you have facilities for TEM, even better.
best wishes for the work.
Ben
Hi Patrice,
I have had some experience with live labeling using lysotracker in cell lines and primary neural cells (adherent cells, imaging using fluorescence widefield microscope). I agree with all the above, very relevant points were brought up. I definitely recommend the references recommended by Sebastian, these were extremely useful for the studies I am presently completing.
I would also suggest considering different manipulations of autophagy using known pharmacological inhibitors and activators as controls. This will help to understand how the autophagic system is working under your specific conditions. For example, Rapamycin and nutrient (amino acid and serum) withdrawal can increase autophagic flux, while inhibitors such as 3-methyladenine, wortmanin, chloroquine, and bafilomycin can reduce autophagic flux. I saw increases in lystotracker stained vesicles (read 'acidic organelle labeling') with 200nM rapamycin (after 24hrs) and serum withdrawal (2-3hrs) and very low or faint staining with agents that inhibit acidification (chloroquine and bafilomycin). Wortmanin and 3methyladenine are a bit tricky because they can exhibit off-target effects as well (I saw increases in staining with 3MA at 5mM)
What is more, timing is very important, and you mention it in your hypothesis stated above. Observing how the staining changes with time is going to be essential. In my experiments, I see both dose- and time-dependent (1-24hrs) responses with the drug I'm using. I saw an increase in lystotracker stained vesicles at low concentrations and then a decrease at higher concentrations where I also observed toxicity. At low concentrations, I saw a consistent increase over 24hrs (1,3,6,12,24h time points).
I hope this information is useful. Again, I gained the most insight about the dynamics of autophagy in my system by using well characterized inhibitors.
Best and happy experiments,
Sebastien
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