PKH26 is a cyanine dye whose structure contains lipophilic groups that allow it to quickly and non-covalently integrate into the cell membranes of almost any cell. This dye is also well-suited for studying membrane vesicles, including exosomes.
Below, we provide a protocol for staining exosomes with PKH26 dye:
1. The PKH26 dye working solution was prepared by adding 9 μL of 96% alcohol to 1 μL of 1 mM PKH26 dye stock solution.
2. The exosome suspension was adjusted to 1 mL using 1× PKH Dyes Diluent.
3. 6 μL of PKH26 dye working solution was added to 1 mL of exosome suspension.
4. The exosome suspension with the dye was carefully pipetted for 30 s and left for 5 min at room temperature in the dark.
5. 2 mL of 10% BSA prepared in 1× PBS was added to the sample.
6. The sample volume was adjusted to 8.5 mL using DMEM.
7. The sample was centrifuged in an ultracentrifuge at 190,000 × g and 2-8 °C for 2 h.
8. Stained exosomes were cleared from the unbound dye. For this, the resulting reddish exosome pellet was resuspended in 1× PBS.
9. The suspension of stained exosomes was transferred to an Amicon MWCO filter column (10 kDa). 9 mL 1× PBS and 0.75 mL culture medium were added.
10. The sample was centrifuged at 3000 × g for 40 min until the volume was 0.5-1 mL.
11. The exosome concentrate was removed from the column and added to a culture of macrophages obtained from human peripheral blood monocytes.
12. Macrophages were cultured with exosomes for 12 h.
13. Visualization and assessment of macrophage uptake of labeled exosomes were performed under an Olympus FW3000 confocal microscope.
Example of the staining:
Assessment of exosome uptake by M2 macrophages using PKH26 dye. The exosome suspension was prestained with PKH26 (x3201) and added to the M2 macrophage culture to assess cellular uptake. Cell nuclei were stained with the nuclear dye Hoechst 33342 (xH010).
Also, additional information on using PKH26 dye to stain membrane vesicles can be found here.