Hi I think you need to specify more precisely what you actually want to quantify. From your question I gather that you want to state something about changes in the early endocytic pathway. Properly employed an estimation of these proteins and an IF characterization is a very good combination. But you need to perform the IF quantification properly, which is depending on a variety factors.

Best regards Andreas Brech

Simply measuring the levels of EEA1 and/or Rab5 can't tell you how many endosomes are present. For this parameter you could use automated microscopy and count spot number and intensity for established endosomal markers such as EEA1, endocytosed Transferrin or a rab protein such as Rab5.

I agree with Matthew and Andreas. Depends on what you really want to quantify.

I would stain normal and treated cells with your markers and then acquire immunofluorescences confocal stacks.

By segmentation is then possible to define the number size etc of the objects.

Transferrin would be ok in the first few minutes of the experiments since it is trafficking pretty rapidly in the late endosomes.

I don't know if I would do it by subcellular fractionation and a gradient. Difficult to see a shift in the population. I don't see the use of nanosight.

e

I also agree with Andreas and Matthew and having nothing further to add.

Paul

Immunofluorescence staining with anti-Rab5 and EEA1 is a quick method, but you should take care because late endosomes/multivesicular bodies usually present some Rab5 and EEA1 in addition to CD63, Rab7, etc... So I also agree that adding a transferrin internalization assay will confirm those early endosomes. Anyway, it depends on your interest, if you can asume that some late endosomes ( probably a little portion) will be marked with EEA1 or Rab5, it is the simpliest way to do it.

Bruno.

Thanks all for your advice. For the ex-vivo experiments I will plan transferrin internalization assays too. For quantification in mice tissue it is a good point to take a closer look also to late endosomes.

I don´t know it is possible to adapt transferrin internalization assay for the use in vivo. I will search in the literature. In my specific case I have to label early endosomes in the pancreas. Any suggestion?

Clara

Hi, today I have a specific question

Hi, today I have a specific question. We performed dextran-internalisation assay (Dextran-biotin 10kDa). Cells were pulsed for 5min, fixed, stained with strep-Alexa488. Representative confocal pictures are attached.

A-ctr

B-postive control, endocytosis is induced by EGF

C,D and E- are my experimental conditions.

My first question is about quantification of endocytic vescicles in these pictures. Due the clear signal/noise ratio I could quantify the total fluorescence and normalize it to the nuclei. This value will be proportional to the number of endosomes. Alternatively, do you think it will more stringent to define a threshold and to count the green vescicle/cell (this will be difficult for exp. 3.). Is it enought to take the median picture, or should I quantify the fluorescence throughout the Z axis?

My second question is the qualitative interpretation of the results: exp.1 shows less/no difference in endocytosis to the ctr. Exp2 and 3 show increase in endocytosis, Exp2 could be very early endosomes due to the localisation near P.M. In exp. 3 there is massive endocytosis (also late endosomes). Is this correct?

Thanks in advance. Clara

One could use endosomal markers and quantify colocalization. Hence quantifying every z section becomes necessity unless if you use Imaris to quantify the whole Z image. Since Exp3 has massive internalization, one could think of increasing the time points between Exp2 and Exp3 and thereby take images that could be quantified. Have you thought of using endocytosis blockers as controls? If so, which one do you use? Best

Narayan