Please, be careful with interpreting Cyto-ID data. I know from my own experience and that of autophagy experts that it is really questionable whether Cyto-ID is specific for autophagy. I would verify with other dyes that stain or track vesicular compartments like lysotracker. 

Greetings, Toine ten Broeke.

Geee, thanks for it, Prof Broeke (the adds say other). Technically, (because of variability of cellular response of autophagy), you should measure blocked, and activated controls in every experiments. That would be the range, where the actual experiment works. That is all, what is described in your second link. I think it is simple so you can give a try.

I use the CytoID to measure autophagy inhibition. This is my experience with it: 

- It won't work in every cell type. 

- measurments are not robust. Always have good controls (10 uM everolimus, 10 mM 3-MA). 

My advice to you: always confirm your results with WB. Perform the experiment with addition of 100ng/ml bafilomycin then do a WB. 

I would'nt block the autophagic flux then add the CytoID, because the agent is supposedly amphiphilic and will accumulate in the autophagosomes based on their pH. When you block the acidification with bafilomycin or other agents, you might change the pH of the autophagosomes and your measurments won't be reliable. 

p.s. why didnt you consider the mcherry-gfp-lc3 construct to measure the flux? 

Thanks for all of your replys!

We're using Leupeptin + NH4Cl as treatment/positive control. For sure we're also double ckeck using LCIII Blots.

The Cyto-ID measurement would be a additional dataset and I thought it's easier to show the Delta (treated/untreated) compared to the full data set.

@Ammar: the GFP-construct would be an easy way but the efficiency is quite low since we're working with iPSC derived neurons.