We performed a dextran-internalisation assay (Dextran-biotin 10kDa). Cells were pulsed for 5min, fixed, and 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 the quantification of endocytic vesicles in these pictures. Due to 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 vesicle/cell (this will be difficult for exp. 3.). Is it enough 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?
Firstly, it appears from your images that your green color is strongly saturated. This will dramatically affect your results (you will underestimate your endocytic efficiency). You need to make sure that your maximum green intensity is well below the maximum image intensity (255 for 8-bit images and 4096 for 12-bit images). Your microscope should have a saturation lookup table so you can assess this.
Secondly, given the non-uniform nature of the staining, you need to collect a z-stack with optimal spacing and the same spacing between samples. Then do a sum projection before summing the signal. Otherwise, you will underestimate early endosomes and overestimate late endosomes (if that is truly what these are). Basically, a single slice contains a far lower fraction of the membrane region than it does of the cytoplasmic region. You will have to be very careful to assign the signal to the appropriate cell for early endosomes. Alternatively, you could sum the whole image, and then divide by the number of observed cells.
As far as counting is concerned, I would doubt that you could count single endosomes for anything other than the control given the density you have. Summing the intensity and interpreting this as endosome quantity would rely on the assumption that each endosome contains the same dextran quantity which may or may not be the case. Alternatively, you could report your findings as "endocytic efficiency" and say nothing about the endosome quantity.
As an alternative approach I will use fluid-phase endocytosis of HRP for the same experimental setup, so I think it will be enough to calculate endocytic efficiency for the dextran uptake.
Dear Clara,
I believe your explanation is correct so for as florscence phenomenon is concern.
Endosome quantification can also be achieved using High Content Analysis. Most manufacturers of these instruments produce software which will identify the cytoplasmic region of cells and then count the number of spots in that image. This gives very good data in relation to receptor internalisation and would also work well with your type of work. An example can be found on our website at www.imagen-biotech.com/AssayExamples/Internalisation.html.
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