Hsp70

To immuno-label exosomes, a previously published protocol was followed (Mondal et al., 2019 (link)). First, the isolated exosomes released from WT or sirt2 KO OLs were permeabilized using 0.001% Triton X-100 for 5 min. Second, PEG10000 was added to the exosomes at a final concentration of 10%, followed by centrifugation at 3,000 g at RT for 5 min. The pellet was dissolved in PBS and incubated with primary antibodies against SIRT2 (ProteinTech, 1:100) and HSP70 (GeneTex, 1:100) overnight at 4°C. Third, 20% PEG10000 was added to the exosome suspension and centrifuged at 3,000 g at RT for 5 min for three times to remove excess antibodies, and then the exosome suspension was incubated with AlexaFluor® 488 and 594-conjugated secondary antibodies (Thermo Fisher Scientific) diluted at 1:100 in PBS for 1 hour at RT. Fourth, the exosome suspension was washed with PEG10000 and centrifuged at 3,000 g three times at RT for 5 min, and passed through Sephadex G-25 column (G2580, Sigma Aldrich) to remove unbound antibodies. Fifth, the exosome pellets were dissolved in 20 μL PBS, placed on microscope slides, covered by a coverslip and kept drying for 5-10 min. Finally, the slides were imaged by a Zeiss 880 confocal microscope with a 1.45 NA 63 × objective.