PKH67 Labeling of Exosome Pellets, a Protocol

PKH67 labeling of exosome pellets, a protocol, By: Michelle Hung Date: 2/16/16

1. Start with exosome pellets (Day 1 – see exosome protocol) 2. Day 2: get the rotor cooling in the ultracentrifuge (see exosome protocol) 3. Combine pellets from multiple tubes into one ultra tube for each sample and measure total volume. 4. Bring your pellet up to 1mL using Diluent C from the PKH67 kit for each sample. 5. Whatever the largest volume exosome pellet was, create another ultracentrifuge tube containing that much exosome free media, and bring it up to 1mL with Diluent C. 6. In microfuge tubes, add 6uL PKH67 in 1mL Diluent C, one tube for each exosome sample, and one tube for the media only control. 7. For each sample, add the dye/diluent mix to the ultra tube. Mix continuously for 30s by gentle pipetting. Quench by adding 2mL 10% BSA in PBS (use the 10x BSA we make for FACS buffer). Then bring the whole thing up to 8.5mL in serum-free media. 8. Make a 0.971 M sucrose solution (I used the protocol described in this paper: http://www.ncbi.nlm.nih.gov/pubmed/22722367.). From a 2.5M Sucrose stock solution (made ahead and stored in the fridge), I make the .971M each fresh each time as it can get contaminated. I add 5.116mL water, 1mL 10xPBS, and 3.884mL sucrose. 9. Add 1.5mL of this solution by pipetting slowly and carefully into the bottom of your tube, making sure not to create turbulence. You should then have your exosomes/pkh67 solution on top of a sucrose cushion. 10. Centrifuge at 192,000g for 2h at 4 degrees. Your exosomes will be in the pellet, and most of the excess dye should be in the interface layer. 11. Carefully aspirate the media and interface layer. 12. Resuspend the exosome pellet by gentle pipetting. 13. Transfer to a centriprep 10kDa cutoff filter column. Add 9mL PBS, 0.75mL media. 14. Spin at 3000g in the high speed centrifuge for 40min, 10min, and as many shorter spins as needed to reduce volume to 0.5-1mL 15. Recover concentrate from centriprep column, store in microfuge on ice. Make a 1/25 dilution in PBS for counting on the NanoSight. 16. Add equal numbers of dye labeled exosomes to cells for uptake analysis.