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After completing the secondary antibody staining at step-5 (in above), unbound secondary antibody from each reaction was washed using PEG10000 as mentioned above. After the 3rd wash, the pellet was dissolved in 50 μl of PKH diluent and 0.2 μl of PKH67 (PKH67GL Sigma) was added to it.
The mixture was incubated at RT for 30 mins with gentle shaking.
50 μl of 20% PEG10000 solution was added to the mixture for the EV precipitation. Then the mixture was centrifuged at 3000 x g for 5 mins. The supernatant was removed very carefully and the EVs were suspended in 50 μl of PBS.
[Note: You may not get a visible pellet here if you begin with less than 20 ml of CM].
The mixture was passed through the column (Sephadex G25). Flow through was collected and 100 μl of 20% PEG10000 was added to it. The mixture was centrifuged at 3000 x g for 10 mins (visible pellet may not be seen). The supernatant was removed very carefully and 10 μl of PBS was added to it.
[Note: Sephadex G25 column is not efficient to remove unbound PKH dye].
Finally, 3–4 μl of dissolved pellet was placed on pre-cleaned slide. A thin smear was drawn as mentioned above (step 8) and mounted for visualization by confocal microscopy (Leica TCS SP5 II).
Next, we checked the integrity of the EVs by transmission electron microscopy (TEM) using gold labeled secondary antibodies.
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