EC isolation with anti-CD31 conjugated Dynabeads

The pellet was resuspended in 100 µl isolation buffer containing 5 µl anti-CD31 conjugated Dynabeads and the suspension was incubated for 15 min at 4 °C with slight rotation. For positive isolation of ECs, 700 µl of isolation buffer was added, the reaction tubes were gently inverted and subsequently placed into the DynaMag-2 magnet for 5 min. The supernatant, containing non-endothelial cells, was removed, and Dynabeads with interacting ECs were subjected to three further washing steps in isolation buffer to remove contaminating cells. Finally, cells were resuspended in 1.5 ml EC-culture medium (M199, 20% FBS, 2.2 g/l sodium bicarbonate, 2.38 g/l HEPES, 100,000 units/l penicillin, 100 mg/l streptomycin, 100 µg/ml heparin, 50 µg/ml endothelial cell growth substrate; Sigma-Aldrich, St. Louis, MO, USA).

For verification of a successful binding of the anti-CD31 antibody to the Dynabeads as well as successful binding of the CD31-conjugated Dynabeads to endothelial cells, 5 µl of the isolated EC suspension can be retained and used as control: The CD31-antibody on the Dynabeads can be visualized by Alexa488 cross-linked goat anti-mouse IgG (Cat. No. A28175, Invitrogen, Karlsruhe, Germany). This results in green-fluorescent Dynabeads, bound to ECs, when observed with fluorescence microscopy. In case fluorescence of the ECs is observed, an excess of anti-CD31 antibody is present and the washing-steps during the anti-CD31-Dynabead conjugation procedure should be increased.