Vesicles were prepared from either JAWS II (ATCC) or Anxa2+/+ and Anxa2−/− dendritic cells. After cell disruption by cavitation in 0.25 M sucrose in DPBS, and homogenization for 10 times with a Dounce homogenizer, the post-nuclear supernatant fraction was centrifuged at 2,000 g for 15 minutes. The supernatant was then centrifuged at 100,000 xg for 1 hour to eliminate residual fragments of plasma membrane, ER, organelles, and Golgi. The fraction of interest, which contained mostly vesicles, was harvested from the supernatant by pelleting at 300,000 xg for 1 hour.
Vesicles were labeled with primary anti-annexin A2 (Clone A-15, Santa Cruz Biotechnology) and anti-Atg16L (Clone AB1, Sigma or Clone 1F12, MBL) antibodies, followed by the secondary goat anti-Alexa-488 and rabbit anti-Texas red-conjugated (Jackson ImmunoResearch) in staining buffer containing 220 mM KCl, 5 mM NaCl, 5 mM NaH2PO4, 0.5 mM MgCl2, pH 6.0. Antibody-stained vesicles were sorted on a Becton Dickinson FACS-Aria high-speed cell sorting flow cytometer. Data acquisition and analysis were performed using BD FACS Diva software.
Vesicles were labeled with primary anti-annexin A2 (Clone A-15, Santa Cruz Biotechnology) and anti-Atg16L (Clone AB1, Sigma or Clone 1F12, MBL) antibodies, followed by the secondary goat anti-Alexa-488 and rabbit anti-Texas red-conjugated (Jackson ImmunoResearch) in staining buffer containing 220 mM KCl, 5 mM NaCl, 5 mM NaH2PO4, 0.5 mM MgCl2, pH 6.0. Antibody-stained vesicles were sorted on a Becton Dickinson FACS-Aria high-speed cell sorting flow cytometer. Data acquisition and analysis were performed using BD FACS Diva software.