Mar 03, 2025
PKH67 Extracellular Vesicle Labeling
- Emily Tabaie1
- 1University of California, Riverside
Protocol Citation: Emily Tabaie 2025. PKH67 Extracellular Vesicle Labeling. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9kmwlk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 03, 2025
Last Modified: March 03, 2025
Protocol Integer ID: 123715
Abstract
This protocol is designed to visualize extracellular vesicles with the lipid dye, PKH67.
PKH67 Extracellular Vesicle Labeling
PKH67 Extracellular Vesicle Labeling
In a microcentrifuge tube add 100 ug (100ul) of EVs in 1 mL of PBS
Centrifuge at 16,100 g for 10 minutes to form a pellet
Discard supernatant without disturbing pell and dissolve the pellet in 100 ul of diluent C and pipette up and down
In a new microcentrifuge tube add 4 ul of PKH67 to 100 ul of Diluent C and Vortex
Combine 100 ul of 2X EV with an equal volume of the pKH67 solution
Pipette the mixture 10 times
Incubate solution at 37°C for 5 minutes at 300 rpm away from light
To stop staining add 400 ul of 1% BSA to the mixture and incubate for a minute for excess dye to bind
Wash stained EVs with PBS 3 times and centrifuge the sample at 16,100 g for 5 minutes
Discard the supernatant and dissolve the pellet in 100 ul of PBS to get a final concentration of 1 ug/ul
Two days before transfer endothelial cells to a poly-L-lysine coated coverslip
Allow the cells to grow a monolayer on the coverslip
After the monolayer is formed removed the medium carefully and add 1 mL of DMEM to wash the cells twice
Add 50 ul of PKH67 stained EVs to the coverslip and incubate for 2 hours
After incubation aspirate the media and wash with PBS 3 times
Add 1 mL of 4% PFA (made in PBS) to coverslips and fix cells for 15 minutes covered in foil away from light shaking
Wash coverslips with 1X PBS 3X for 1 min each wash
Add 1 mL to each well of Perm buffer (Triton X, BSA, PBS) to permeabilize the membrane on the shaker for 10 minutes shaking
Aspirate the Perm Buffer and wash coverslips with 1X PBS 3X for 1 min each wash
Block with 1 mL of 5% Donkey Serum (DS) diluted in 1X PBS and incubate on the shaker for 30 min at room temperature shaking
Aspirate and add 800 ul Primary Antibody Staining in Blocking Buffer and incubate for 1 hour at room temperature shaking
Wash off unbound antibodies 3X with 1X PBS for 1 min each wash
Aspirate and add 800 ul of Secondary Antibody Staining in Blocking Buffer and incubate for 1 hour at room temperature shaking covered in foil
Wash off unbound antibodies with 1X PBS 3 x 10 min each wash shaking
Grab microscope slides and label them with the experiment, Ab used, initials, and date
Add (a small) drop of DAPI mounting media for every coverslip, and mount on glass slide
Cure slides overnight and image