Anti gpc1 antibody

One milligram of exosomes were incubated with 300 μl of aldehyde/sulphate latex beads (Invitrogen Inc., Carlsbad, CA, USA) for 20 min. at RT with continuous rotation. After dilution with 1× PBS and stopping reaction with 100 mM glycine and 2% bovine serum albumin (BSA) in PBS for 30 min. at RT, exosomes‐bound beads were washed once in 1× PBS containing 2% BSA and centrifuged for 1 min. at 14,800 × g and blocked with 10% BSA for 30 min. The re‐suspended beads were incubated with anti‐GPC1 antibody (Santa Cruz) for 30 min. at 4°C with rotation. After centrifugation, washing and re‐suspension, beads were incubated with Alexa‐488‐tagged secondary antibodies (Life Technologies, Carlsbad, CA, USA) for 30 min. at 4°C with rotation. Secondary antibody incubation alone was used as control and to gate the beads with GPC1‐bound exosomes. The percentage of beads with GPC1⁺ exosomes was calculated.

We collected 20 ml of peripheral fasting blood samples into EDTA tubes and centrifuged at 3000 × g for 5 minutes at 4°C. The plasma was collected and immediately frozen at −80°C. Exosomes were isolated from plasma using ExoCapTM Exosome Isolation and Enrichment kit (JSR Micro Materials Innovation, Sunnyvale, CA, USA) by following the manufacturer's protocol. The isolated exosomes were purified using sucrose density gradients as previously described [12 (link)]. The percentage of GPC1⁺ plasma exosomes was determined by flow cytometry. Briefly, one mg of exosomes was incubated with 300 μl of aldehyde/sulphate latex beads (Invitrogen Inc., Carlsbad, CA, USA) for 20 min at room temperature. The reaction was stopped with 100 mM glycine and 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 min at room temperature. The exosomes-bound beads were then washed with 1 × PBS containing 2% BSA and centrifuged for one min at 14,800g. The re-suspended beads were incubated with anti-GPC1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min, followed by incubation with Alexa-488-tagged secondary antibodies (Life Technologies, Carsbad, CA, USA) for 30 min at 4°C with rotation. Secondary antibody incubation alone was used as control and to gate the beads with GPC1-bound exosomes. The percentage of beads with GPC1⁺ exosomes was calculated.