Anti cd45

pMSC were characterised by flow cytometry using anti-porcine specific or cross-reactive antibodies as previously described [28 (link)]. In particular, anti-CD45 (LifeSpan Biosciences, Seattle, Washington), CD29 (Acris Antibodies, Herford, Germany), anti-CD90, anti-CD11b and anti-CD105 (all from Abcam, Cambridge, UK) were used. Cells were tested for their ability to differentiate into osteoblasts and adipocytes at early passages (P3), as previously described [28 (link)].

Microvesicle and exosome fractions from 16 ml urine samples were separated by SDS-PAGE (8–16% acrylamide gradient) and then transferred to a nitrocellulose membrane. The membrane was blocked with 1% bovine serum albumin (BSA) in PBS plus 0.1% Tween-20 (PBST), rinsed in PBST, and labeled with one of the following primary human antibodies diluted in 1% BSA in PBST: anti-CD63 (Novus Biological, Littleton, CA, USA), anti-CD81 (Novus Biological, Littleton, CA, USA), or anti-CD45 (LifeSpan BioSciences, Seattle, WA, USA) [41 (link)]. After rinsing again in PBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (Novus Biological, Littleton, CA, USA). Chemiluminescence was detected and quantified using the ChemiDoc Touch Imaging System and Image Lab software (Bio-Rad, Hercules, CA, USA).