Suggestion:

You can have a phenomenon of "caping.

When it makes a fluorescent labeling, is sometimes seen a migration of antigens in the membrane, they focus and one gets a point of fluorescence (caping)

I frequently watched him on some labeled cells (K562, HL60, ...). As the Protocol is the same and that I was not watching it for all the cells marked (and positive), it seems to be 'random' but I don’t know the mechanism of this phenomenon

On the other hand, in some cases, when it stimulates membrane-bound receptors they can regroup (caping)

I am virtually on the existence of these phenomena but I don’t have citations to offer

Sorry

Best regards

Philippe

Thank you Phillippe,

However, LC3 is an intracellular molecules. Could that phenomenon happen to those molecule?

Thank you again!

When talking about LC3 as marker for autophagy, you have to keep in mind that it has 2 forms, LC3-I and LC3-II. LC3-II is formed by lipidation of LC3-I and is the marker for ongoing autophagy. Going to Western blots, you will find LC3-I at a molecular level of approx. 16-17 kDa and LC3-II at approx. 14-15 kDa. During autophagy LC3-II level will increase whereas LC3-I level will decrease.

LC3-I is a cytosolic form of the LC3 protein whereas LC3-phosphatidylethanolamine conjugate (LC3-II) is recruited to autophagosomal membranes.

Thank you Bruennert,

I did western blot already and now I would like to observe autophagy event by immunofluorescence. It's my very first time so I was confused to determine whether the things that I saw are autophagosom or not...

Do you have any idea about my photos?

Thank you again!

I never did the LC3 staining on my own, still have to do this job. But I am pretty sure that LC3 is staining the autophagosomal vacuoles, so the autophagosomes which engulf cytoplasmic components. And I would not be surprised to find LC3 marker in both spherical vacuoles in the perinuclear region of the cells, as well as in foci distributed throughout thecytoplasm. According to what I found in literature, the distribution of LC3 seems to be dependent on the cell type as well as probably the intensity of autophagy induced by different stimuli.

In fluorescence study if LC3 has been labeled properly then your results are indicating an increase in autophagy level. But to be confirm about it next time use a +ve control by treating cells with rapamycin. And to count the no of autophagic vacuoles take Z-sections and count no of dots per cells.

Personally, I don't think that Rapamycin is a good autophagy-inducer. Chloroquine is a much better autophagy-inducer than Rapamycin - and a combination of Chloroquine and Rapamycin gives strongest autophagy-results.

When you go to FACS or immunofluorescence staining pictures that you find in the autophagy kit handbook from Enzo (Cyto-ID® Autophagy Detection Kit

Catalog No. ENZ-51031), you can see how weak the autophagy induction after Rapamycin is compared to Chloroquine.

I am a beginner in this field. I found in literature that chloroquine is an inhibitor of autophagy. When doing immunofluorescence in chloroquine treated cells, chloroquine inhibits lysosome fusion and as a result autophagosomes accumulate. Whether this autophagosome accumulation can be said as induction of autophagy?

I would add another control in your experiments. It's very important to keep in mind that almost all cell type has basal autophagy. So, I will BLOCK the autophagy flux first in untreated cells (you can use protease inhibitors, bafilomycin or other compound). Other control would be traeted cells with an autophagy inducer like Rapamycin plus the same compound used to block the flux. Finally, your conditions must carry on the same blocking flux drug. The point is to compare the basal state with the blockade flux with your conditions with the flux blocked. So, you could see how increased is the autophagy in your conditions. 

Regards,

Javi

P.S: In the link attached you will find both, western blot and immunofluorescence assays.

http://www.jbc.org/content/early/2013/09/06/jbc.M113.490581

Chloroquine will block the degradation of autophagosomes but not not induce autophagy. In order to induce autophagy you have to use a compound like Rapamycin or starve cells with HBSS - maybe try both methods, since Rapamycin does not always work. You might want to combine this with your treatment to see if your treatment increases HBSS or Rapamycin induced autophagy. Also, check if the fusion with the lysosome is affected. For this use either a tandem construct ( dsRed/RFP - GFP LC3) or label lysosomes for marker proteins. In addition, look at p62/SQSTM1 levels. Personally I am sceptical about using flow cytometry, and it seems not be used very often for this type of experiments.

Thank you all for your helpful suggestion.

I will add the control cells with autophagy inhibitors to see the difference in morphology of the LC3 puncta.

Good ...

In the fig 2(down) appears that there is some indication of autophagy indution since the Puncta formation of LC 3 was observed. Further I want to see the higer magnification of these images to predict clearly.

Did you do any tratment for the  primary blood neutrophils for autophagy induction?

Dear Duy,

As I look into your figures I would say that your cells induced autophagy after treatment. I wouldn't say much about the morphology because you compare here a basal with a treated condition, but an increase in autophagosomes number is definitely clear. In western blot, could you also detect the increase of LC3-II and p62 in your cells after treatment?

I agree with Javier that you should add autophagy inhibitor (bafilomycin, chloroquine) to stop the flux, I wouldn't use autophagy inducers (Rapamycin, Torin, starvation) here because it seems like that the drug you used to treat the cells could already an autophagy inducer.

I would design the experiment like:

Condition 1: - Drug / - Inbibitor

Condition 2: - Drug / + Inhibitor

Condition 3: + Drug / - Inhibitor

Condition 4: + Drug / + Inhibitor

and firsly run the western blot to detect any changes in protein level of LC3-II and p62. If I could achieve any significant results, then I would immunostain the cells with LC3-II to validate the blot results.

I hope my comment would help you a bit.

Regards,

Duy

Hello Duy,

Thank you for your helpful suggestions.

I did perform western blot to detect LC3-II band. It worked well. Then I moved on investigating autophagy by ICC.

However, in western blot, I could not detect p62 in neutrophils. I tried to use different antibodies, different conditions but got nothing.  Do you have any idea?

Moreover, I do not know which method, determination of autophagy positive cells (which have more than 10 LC3 puncta) or the counting the average number of LC3 puncta in each cells, is better for quantifying autophagy flux in neutrophil by ICC. Do you have any suggestion?

Thanks again and warm regards,

Duy.

I have a question. Since LC3 has 2 forms, how do you know it is LC3-II but not LC3-I in the immunofluorescence picture?

Dear Chao, 

If you observe immunofluorescent dots when stained for LC3 it is considered that the dots represent LC3 in autophagosome, hence it is LC3II. If it is spread around the cell cytoplasm it's LC3I. 

To put it simple:

dots = LC3II

Good luck

Hi Chao,

The thing we want to investigate is the autophagosome. In the mechanisms of autophagosome formation, LC3-I is converted into LC3-II and bind to the membrane of autophagosome. Therefore, in western blot, we detect LC3-II as an indirect marker for autophagosome formation.

However, when we use imaging methods (TEM, confocal microscopy), we can observe directly the autophagosome, and there is no need to look at LC3-I or LC3-II.

Hope this helps.

Duy

Actually ( your LC3B punctuate be distributed in cytosole and localized in nucleus as well . as in image LC3B m2a and you can count the autophagosomes per cell in one image . or autophagosomes count per cell (3 cells ) per one photo ( if you took 5 photos ) for each condition you can have a nice graph showing error bars and significance of your results .

Help this would help

you can count the autophagsomes using cell counter plugin on FiJi .