Hi Sonja

As you say there are several variations to the protocol for this assay and I have been trying several so I will try to answer some of your questions and tell you a bit about my personal experience with this assay.

The two variations you mention with TCA are similar to the changes in the assay I do depedent of whether I am working or adherent or suspension cells. I have had success with the assay with scraping adherent cells into TCA or adding TCA directly to suspension cells so I think both should work and I think they give the same outcome. From what I have been able to find, TCA does lyse the cells. In the protocol you quote, I understand it as that they do not scrape the cells and use TCA to lyse them. I have not tried this myself, but I think it would be equivalent to scraping into TCA.

Regarding the inhibitors, I guess it can be fine to do an initial approach with just complete media, starvation media and starvation with your compounds to see if there is an effect on autophagy. However, if you do see an effect, it can be very useful to include inhibitors such a 3-MA and BafilomycinA1 together with your compounds and separately to give additional information.

I have also been experimenting with extended times of radioactivity incorporation (up to 3 days) and longer times of short-lived protein chase. I have found that longer times of incorporation and longer times of chase (up to 16h) gives a stronger starvation-induced signal and less background from short-lived proteins, but shorter times of both incorporation and chase also work fine.

Hi,

thank your for your answer! So what kind of protocol you are usually using for adherent cells? Do you TCA-precipitate the medium separately or do you scrape the cells into media and then TCA-precipitate the whole cell suspension? I think the first method decreases the amount of sample tubes but is it as reliable?

Klionsky's paper clarified the use of 3-MA in this case - it can be helped to separate other degradation mechanisms from macroautophagy.

I perform this assay in 24-well plates to minimize costs and the time spent. Then I do not scrape at all, but rather just move the media into TCA. I wash the cells once in 0.1 g/ml BSA and also add that to the TCA then add 0.2M strong base to the cells in the well.

After centrifuging the media, I move the supernatant to counting vials and dissolve the pellet in base and add that to the wells then move the well content to counting vials.

This variation is slightly different from some others in that any acid-soluble radioactivity inside the cells will be counted in the well fraction, as compared to a TCA lysis protocol where it would be released and counted with the supernatant fraction. I have compared my variation with a scraping-into-TCA protocol and in my setup the protocols gave identical results, suggesting that the amounts of acid-soluble radioactivity in the cells is similar (and possibly relatively small) independent of autophagy-affecting treatments.

After reading through the protocol you linked, and assuming that TCA does in fact lyse the cells, I think it might actually be even better to just add TCA to adherent cells as in that protocol because then intracellular acid-soluble radioactivity should also be included in the supernatant. Let me know if you try this, as I would be interested to hear how it goes. Perhaps anyone else has any experience with TCA lysing (or not lysing) cells?

Thank you for the clarification!

I'll probably try the protocol in which cells are scraped into medium, wells are washed with BSA-containing EBSS and the whole suspension is TCA-precipitated. That method is used in this paper: https://www.landesbioscience.com/journals/autophagy/2012AUTO0209R2.pdf

And I'd be glad to hear how other people are performing this assay. :)

Hellow there

As I found you want to find the best way to measure a long-lived protein degradation.

I agree with Petter, However, I suggest you remove all media from the well and keep it in a separetely vials, then wash the cells with PBS 3 times. In this step, scrape the cells. Now you have two vials, supernatant and cell which you can resuspend it in the PBS.

for degredation assay you can investigated the both supernatant and cell suspension in the SDS-PAGE Gel Electrophoresis.

In addition you can measure your protein after 24 h of incubation the cells and lyse them with a suitable lysis buffer, so that you have the early amount of protein. In this regard you can spectrophotometery measured the amount of protein which has been degredated.

I hope I Understand your question correctly.