Anti cd45 magnetic beads

Placental EVs were collected from human placentae using a well-established ex vivo placental explant culture method as previously described (28 (link), 30 (link)). Briefly, 400 mg explants were dissected from the villous placenta and cultured in Netwell^TM inserts in Advanced DMEM/F12 medium supplemented with 2% FBS and 1% Penicillin/Streptomycin (Invitrogen). After 18 h of culture at 37°C in 5% CO₂/95% air, the culture medium was aspirated and placental EVs of different sizes were separated by sequential centrifugation at 4°C at 2,000 g for 5 min (macro-vesicle fraction), 20,000 g for 1 h (micro-vesicle fraction), and 100,000 g for 1 h (nano-vesicle fraction) (Avanti J30I Ultracentrifuge, JA 30.50 Ti fixed angle rotor, Beckman Coulter). Contaminating red blood cells were removed from the macro-vesicle fraction by hypotonic lysis in ultrapure water (EMD Millipore) and contaminating leukocytes were depleted using anti-CD45 magnetic beads (Dako).

Placental EVs were collected from cultured human placentae as previously described^{4 (link),12 (link)}. Briefly, 400 mg explants were dissected from the villous placenta and cultured in Netwell^TM inserts (Corning) in Advanced DMEM/F12 medium supplemented with 2% FBS and 1% Penicillin/Streptomycin (Invitrogen). In some experiments, ID2 (50 μg/mL), isotype-matched control antibody (50 μg/mL), patient aPL (50 μg/mL), control IgG (50 μg/mL), or a fluorescent dye CellTracker® Green CMFDA (2 µg/mL, Invitrogen) was added into the culture medium. After 18 hours of culture at 37 ^oC with 5% CO₂/95% air, the culture medium was aspirated and placental EVs of different sizes was separated by sequential centrifugation at 4 ^oC at 2,000 g for five minutes (macro-vesicles), 20,000 g for one hour (micro-vesicles) and 100,000 g for one hour (nano-vesicles) (Avanti J30I Ultracentrifuge, JA 30.50 Ti fixed angle rotor, Beckman Coulter). Contaminating red blood cells were removed from the macro-vesicle fraction by hypotonic lysis in ultrapure water (EMD Millipore) and contaminating leukocytes were depleted using anti-CD45 magnetic beads (Dako) per the manufacturer’s instructions. The physical characteristics of these different sized vesicles has recently been extensively characterised^{4 (link)}.