Hi! Phalloidin stains actin, so the cytoskeleton, it comes to ereason that if there is co-localization then it is indeed in the cell.

I stain with phalloidin all the time, I buy the green (from dyomics, germany) variation and dilute 1:500 and it is pretty straight forward, fix your cells (phalloidin only works in fixed cells) I make a solution of Phalloidin plus Hoescht, stain for one hour, wash once with PBS and image! It is super simple and very rarely do I have any issues with this technique. You can also go with a membrane dye to insure that there is no membrane accumulation, I have used this before and bought from Promokine, Germany, check their website there are many options

Hi Felix,

if you want to stain actin I can highly recommend Alexa Fluor conjugated Phalloidin by Invitrogen. Gives you a superb staining and no background.

Would try rab7 for late endosome, rab11 for recycling endosomes. Also should mark Golgi ( giantin, golgin97 ) and ER. Would do 3d rendering on stacks and look for voxel overlap with your protein localization with the organelle markers. Plus if you label the cell membrane and do a 3D rendering you fluorescent signal from your uptake will be inside the shell formed by membrane staining.

The best way to stain actin is phalloidin conjugates.To localize the opical level of your microscope u should additional stain the nucleus with DAPI or equal. Then do a quick scan and adjust the optical axis so that the nucleus is visible. Afterwards perform a normal detailed scan of ur cells.

I am not working with HeLa cells myself, but if you think that your protein is located in an acidic compartment within the cytosol (e.g. late endosomes) you could try the LysoSensor or LysoTracker dyes.

Hi Felix,

I would suggest you look at lysosomal co-localization as well since that is often the fate of large endocytosed objects. Labeled LDL (DiI-LDL is common) is a good marker for lysosomes. You can probably use this in tandem with your commercial early endosomal markers. Another option is one of the Lysotracker dyes that will stain all acidic compartments (later endosomes + lysosomes).

If you are convinced that your particle is escaping the endosomal system and getting into the cytosol, perhaps a cytosolic tracker will be more useful than the actin-binding phalloidins. Unless there is a direct association between your particles and actin, you may be left with no information on the rest of the cytosolic components (other than actin). Low levels of a cytosolic tracer like calcein AM would stain the cytoplasm though you may have to play with the exact loading concentration to avoid lacquering your cells with fluorophore. Good luck

First of all, I don't think you will have trouble claiming the entry of your particle as long as you viewed your samples with a confocal microscope and it is not accumulating on the plasma membrane. One way to do it is to co-incubate with FM1-43 from Invitrogen, which will label the plasma membrane and endocytosed membrane structure.

Have you examined other endosomal compartment? You mentioned you examined early endosome and potentially late endosome/lysosome with dextran. Please keep in mind that dextran doesn't reach late endosome/lysosome until a good 45 min in most cells, and the endocytic rate of different ligand may vary also. The best way to analyze this is to do a pulse chase experiment with your ligand. Fix and co-stain with early endosome markers like (EEA1, or the CellLight marker you mentioned) and late endosome/lysosome with lysotracker all together for every 10-15 min.

You might want to check co-staining with Golgi (especially TGN) and endocytic recylcing compartment (ERC), in case if your ligand is rapidly recycled back to the plasma mebrane with some unknown receptors. The easiest way to examine ERC is to coincubate your ligands with fluorescent labeled transferrin. You can get it either pre-labeled from companies like Invitrogen or you can get it from Sigma and label it yourself. Hope this help.

You can use FRAP and see the fluorescence recovery dynamics, it will give you an idea of how fast your particles diffuse, if they are bound or not to teh cytoskeleton or to an organelle.

Hello Felix,

Unless your particule distribution is very uniform in the cell, actin staining may not be the best choice to find a cytosolic colocalization with your particles.

Why not transfecting your cells with a plasmid encoding for cytosolic GFP. Then you incubate your cells with fluorescent transferrin as a positive control for endocytosis and finally you can look for colocalization with your particles.

If your particle is colocalized with transferrin you are in endosomes, if colocalized with GFP you are in the cytosol !

Whoa! There are so many good answers that I have almost nothing to add. Some have mentioned time. A lot of cell biologist forget that time applies to cells as well.

You have the chance to use cell culture cells, so you can transfect them. As already mentioned you can use GFP or derivates fusion proteins of markers of cell membrane, early endosomes, late endosomes. But if your particles are escaping the endosomal system and it is using the retrograde transport, you will need ER and Golgi markers. A clue (although it is not very reliable) for use of retrograde transport is the time that the particle needs to reach the final point. If you see after 1,5 h or 2 h your particles accumulated in a structure close to the nucleus, it can be the retrograde transport.

If you just want to show someone that your particles are inside the cell, the methods already described using membrane markers as FM 1-64 or cytoskeleton staining can help you but only after you have done a 3D projection of a Z-stack of your confocal images. For this, ImageJ es an excellent alternative to expensive imaging software. Just make sure that your images are not oversaturated. I wish you good luck.

I do not wish to be a pain, but I really need to point out that we do not "prove" anything in science - we provide evidence for a hypothesis - sorry, my pet peeve!

Hi Fellix,

I would like to add some more points. Rab5 is also a good choice to mark early endosomes besides EEA1, for late endosomes as per above suggestion rab7 will be a good choice. You can try DQ-BSA to stain lysosomes as they are more specific than lysotracker dyes and you can also look for other marker like GM130 (for golgi) or LC3 for (Autophagosomes, as many of the cytosolic particle end up in autophagosomes), for actin I have used Alexa488 and 546 labelled phalloidin and they works very well in Hela Cells. Hope it will help.

Pawan

you could simply image the cells, whilst applying detergent (mild conditions to open the PM) - different concentration of digitonin will keep nucleus intact and affect different cytosolic membranes based on their cholesterol content. Triton X-14, if I remember correctly, also has diverse effects on dissolving the membranes of small vesicles.

Another option would be to check using electron microscopy, especially if your fluorescence-labeled particles are electron dense (at least after preparation for EM). In EM you can nicely see where membranes are and check, if your particles are enclosed or not.

If your particles are rather large you can even try to use FRET to see, if they are incorporated in lysosomes, e.g. like published by Yates et al 2005: http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0854.2005.00284.x/pdf

In this case FRET occurs, because there is no space left, because the beads are so large.