Digital site ds u2

Cells were seeded onto microscope cover glass. After culturing overnight, culture media containing 10% pooled human serum or heat-inactivated human serum was treated for 1 h. The cells were fixed with 4% PFA in PBS and blocked with 3% bovine serum albumin. Cells were incubated with rabbit polyclonal anti-C5b-9 (1:500, Abcam) overnight at 4 °C, followed by incubating with Alexa Fluor 588-labeled secondary antibodies (Invitrogen, Carlsbad, CA). After washing, nuclei were stained using 4,6-diamidino-2-phenylindole and mounted in Vectashield® (Vector Laboratories Inc., Burlingame, CA). Images were observed under the Nikon Eclipse E400 microscope (Nikon Instruments Inc., Melville, NY) using Nikon Digital site DS-U2, and analyzed using NIS element F.

EVs were stained with ExoGlow-membrane EV labeling kit (System Biosciences, Pala Alto, CA, USA) according to the manufacturer’s instructions. After labeling, the EVs were mixed with 10 ml of PBS and the EVs were isolated again by centrifugation at 100,000× g for 1 h. Cells were seeded onto a microscope cover glass. After culturing overnight, the culture media containing the fluorescence-labelled EVs were treated for 3~24 h. The cells were fixed with 4% paraformaldehyde in PBS. After washing, the nuclei were stained using 4,6-diamidino-2-phenylindole and mounted in Vectashield^® (Vector Laboratories Inc., Burlingame, CA, USA). Images were observed under the Nikon Eclipse E400 microscope (Nikon Instruments Inc., Melville, NY, USA) using a Nikon Digital site DS-U2 (Nikon Instruments Inc.), and analyzed using NIS element F (version 4.6, Nikon Instruments Inc.).