Anti cd44

Porcine cATMSC-EV were identified by bead-based flow cytometry, screening for EV and MSC markers. EV were covalently coupled to 4-μm aldehyde/sulphate-latex beads (Invitrogen-ThermoFisher Scientific) with a 15 min incubation, and then blocked for 2 h with BCB buffer (PBS, 0.1% BSA, and 0.01% sodium azide (NaN₃); Sigma Aldrich). EV-coupled beads were centrifuged at 2000×g for 10 min and re-suspended in BCB buffer. Next, beads were incubated for 30 min at RT with the fluorochrome-conjugated antibodies anti-CD73-PE and anti-CD90-PE-Cy7 (1:50; both from BD); or the primary Ab anti-CD9 (Clone VJ1/20; 1:10), anti-CD63 (Clone TEA3/18; 1:10), anti-CD81 (Clone 5A6; 1:10), anti-CD29 (1:10; BD), anti-CD44 (1:10; AbD Serotec) or IgG isotype control (1:10; Abcam) followed by incubation with the FITC-goat F(ab')2 anti-mouse IgG (1:10; Bionova) or A488-rabbit anti-rat IgG (1:100; AbD Serotec). EV-coupled beads were washed with BCB buffer and spun down at 2000×g for 10 min after each step. Data was acquired in a FACSVerse flow cytometer (BD), and analysed with FlowJo® v10 (BD).

The following antibodies were used in the experiments: anti‐CD44 (AbD Serotec); anti‐Sox9 and anti‐K19 (Santa Cruz Biotechnology), anti‐F4/80 (Caltag), anti‐Ki67 (Gene Tex), anti‐HNF4α (Abcam), and anti‐RFP (Rockland).