Hi Venkata,

Cellmask™ Orange is added to the vesicles at a final concentration of 10μg/mL and incubated at 37°C for 30minutes. Excess dye is then removed by washing the vesicles in PBS by high speed centrifugation or using an 30kDa MWCO centrifugal filter (e.g. Amicon). I prefer the centrifugal filter as centrifugation tends to cause EV aggregation.

The diluent C contains NaCl, Tris-HCl, EDTA, DTT, Triton X-100, BSA, glycerol. EDTA somewhat destabilizes plasma membrane. Triton X also increase permeability of the membrane. I assume, as it contains detergent, therefore it will hamper the membrane integrity (to some extent,/ if kept for long time then it can damage the exosome membrane). PKH67 does not react and/or bind with PBS, so no or very less background fluorescence is expected. Serum proteins have binding sites for many dyes. BSA is used to bind excess dye after staining time. Furthermore Serum contains high amount of exosomes so those exosomes also get stained.  

I am not a big fan of using PKH67 in functional assays as the PKH67 may leak from the exosomes and has been shown to be taken up by formalin-fixed cells. Labelling with Cellmask membrane dyes is more stable in  my experience and does not adversely affect the exosomes in functional experiments.

Thank you Suman and Chris for your answers!

@Suman

The Diluent C you mentioned sounds like the NEB Diluent C for diluting restriction enzymes (https://www.neb.com/products/b8003-diluent-c#tabselect0).

Is the NEB Diluent C the same as the Sigma Diluent C? Both have different applications, the NEB to dilute restriction enzymes and the Sigma to dissolve dye for membrane labelling.

@Suman and Navind-

I really doubt that the Diluent C for PKH labelling is the same as the NEB diluent C. 

From this webpage, about Diluent C for membrane labelling (with CellVue, but assume is the same as PKH): http://www.polysciences.com/default/catalog-products/diluent-c/

"Diluent C is an iso-osmotic aqueous solution which contains no physiologic salts or buffers, detergents, or organic solvents"

Therefore Diluent C for PKH labelling wouldn't contain NaCl nor Triton-X, as listed above and on NEB website.

Also the presence of free protein in the form of BSA would interfere with PKH labelling.

Going back to the original patent for PKH dyes- http://www.google.co.uk/patents/US4762701 - I think that the diluent must be either iso-osmotic mannitol, or iso-osmotic glucose. This makes sense, as it's quite viscous.

Hi Kishore,

I typically use the exosomes immediately after labeling, but I have used them a few days after labeling and achieved the same results with cell culture imaging.

@Chris Gardiner...

I have been using PKH dyes for labelling exosomes till now. I would like to try your recipie using CellMask dyes. Could you please provide me the protocol if there is any?. 

Thank you very much for your help

Hi Venkata,

Cellmask™ Orange is added to the vesicles at a final concentration of 10μg/mL and incubated at 37°C for 30minutes. Excess dye is then removed by washing the vesicles in PBS by high speed centrifugation or using an 30kDa MWCO centrifugal filter (e.g. Amicon). I prefer the centrifugal filter as centrifugation tends to cause EV aggregation.

Hello chris... Thank you very much for your quick reply .  I will give it a shot and keep you posted :)