I have problem with somes markers...
Did lysostracker Green or red only stain lysosomes.
Or did this marker also stain acidic compartement as the AO did.
I feel that AO staing is very efficient to follow acidic compartment in normal cells and cells wich are undergoing autophagy until the autopagosomes are excreted or destroy. But this dye stain acidic compatment and we can believe to have a big acidic compartment when lysosomes and autophagosomes are fused.
Did I am right..?
If I would like to trace mitochondria, lysosomes and autophagolysosmes what can I do?
Any help is wellcome
Patrice
It sounds logical to me that you can follow lysosomes and autophagosomes fused with lysosomes both with acidic indicator. The question is, if you want to separately track them, can you discriminate by size. Maybe you could transilluminate the cells with polarized light and use a polarized (90dergees rotated) filter to acquire images to enhance membrane contrast. Then using ImageJ particle size analysis you can track the different sized vesicles.
Another approach is to stain lysosomes with Lysotracker and subsequently immunostain mitochondria via COXIV and autophagosomes via LC3-II labeling. In the attachment you will find details on this technique. I hope this information is useful to you.
Regards, Christian
Article Parallel damage in mitochondrial and lysosomal compartments ...
Thank you Christian for citing our work and trusting our group. I would like to mention to Patrice that one can use immune staining by LAMP2 (glycoprotein present in lysosomes) and LC3II (sequestered to autophagosome). Pure lysosomes will be mainly stained by LAMP2 and autophagolysosmes will have both LAMP2 and LC3II staining. Waleska also used this technique in the paper cited by Christian.
Hi Patrice!
Yes the recent paper from Dr. Baptista's lab could be a very helpful guide. I do have some questions about Fig. 2C and LC3-II staining, I'm confused if it is hoechst and why it is absent in CT LC3-II channel but then appears in CT merged? Any clarifications would be most appreciated.
From what i've heard it is quite challenging to do direct immunostaining of cells against LC3 so people have resorted to generating cells with GFP-LC3 to monitor autophagy. I'm not sure how specific LAMP1 is to the lysosome but maybe you can read on this too. Best wishes!
-ED
Good morning Patrice,
I would agree with Christian. The differential staining of the lysosome and mitochondria would work nicely. The referenced paper from Dr. Baptista's lab sets out what to do beautifully. Very interesting work, Dr. Baptista!
Below with the help of Waleska we answer the questions by Emmanuel:
Q:I do have some questions about Fig. 2C and LC3-II staining, I'm confused if it is hoechst and why it is absent in CT LC3-II channel but then appears in CT merged? Any clarifications would be most appreciated.
R:The nuclear staining with DAPI was only showed in merged panel for all treatments, including control (CT).We chose this type of presentantion of data to clarify the puncta localization per cell that was highlighted by nuclear staining.
Q:From what i've heard it is quite challenging to do direct immunostaining of cells against LC3 so people have resorted to generating cells with GFP-LC3 to monitor autophagy.
R:This question was discussed along the paper, please seee page 3:
"........ immortalized human skin keratinocytes (HaCaT)8, in which homeostasis strictly depends on autophagy pathway9. Consequently, in HaCaT it is possible to detect endogenous LC3 lipidated form (LC3-II)10, avoiding artifacts that may occur when employing transfection and transgenesis strategies11".
Q:I'm not sure how specific LAMP1 is to the lysosome but maybe you can read on this too.
R:LAMP1 is a lysosomal-associatedmembrane protein 1 that usually used as lysosome marker as sugested by several guidelines to monitor autophagy, including the 3rd edition Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Klionsky et al. Autophagy 2016.
Dear Mauricio,
I try to contactc you by meil and get no answer...could you provide me wirh a E-mail adress please.
Sure, [email protected]
Please send again the e-mail. I just checked my mail box and found nothing. I may have deleted inadvertently.
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