Pierce bcatm protein assay

Exosomes were isolated from the culture medium of 8 × 10⁶ ASCs. The cells were cultured to confluence and, to avoid any contamination of shed membrane fragments and vesicles from serum, 48 h of FBS deprivation was performed. Cell culture supernatants were then collected and PureExo Exosome isolation kit (101Bio, CA, United States) was used for exosomes isolation, following the manufacturer’s protocol. Exosomes were resuspended in 100 μl of PBS and used directly or stored at −80°C. The determination of the protein content of exosomes was performed by Bicinchoninic Protein Assay (BCA method) using the manufacturer’s protocol (Thermo Fisher Scientific^TM Pierce^TM BCA^TM Protein Assay).
The treatment with ASCs-exosomes was performed on G93ADOXY+ cells. The cells were seeded and incubated for 3 h with or without ASCs-exosomes (EXO) at a final concentration of 0.2 μg/ml in the culture medium. Control cells were treated with the same amount of PBS.

Co-IP was performed using the Protein A/G PLUS-Agarose Immunoprecipitation Kit (Santa Cruz Biotechnology, USA) according to the manufacturer’s instructions. Briefly, 4× 10⁷ PBMCs were collected from experimentally infected goats (in vivo) were pelleted and lysed with 3 mL of NP-40 lysis buffer (50 mM Tris pH 7.4, 150mM NaCl, 1% NP-40) containing protease inhibitor cocktail (Merck, USA). Cellular debris was pelleted by centrifugation at 10,000 x g for 10 min at 4°C, and the supernatant was transferred to a new tube. The cell lysate was precleared by incubation with 1 μg of rat normal IgG and 20 μL of Protein A/G PLUS-Agarose beads at 4°C for 30 min. After pelleting the beads by centrifugation at 1,000 ×g for 5 min at 4°C, the protein concentration of the supernatant (cell lysate for IP) was determined using the Pierce^TM BCA^TM Protein Assay (Thermo Fisher Scientific, USA).
A 1-mL aliquot of the above lysate was incubated with IgG_HcESP overnight at 4°C. Immune complexes were isolated using 20 μL of protein A/G plus agarose. Immunoprecipitates were collected by centrifugation at 2,500 rpm for 5 min at 4°C. The supernatant was carefully aspirated and discarded, and the pellet was washed four times with RIPA buffer. After the final wash, the pellet was resuspended in 1X SDS buffer.

Enzyme activity was measured in heparinized plasma, buffered 1:2 in 0.2 M Tris, 1.6 mM MgCl₂, pH 8.1 by a previously described colorimetric assay using the synthetic substrate p-nitrophenylthymidine 5’-monophosphate (PNTM) (Rutsch et al., 2003 (link)). Protein concentration was determined using Pierce^TM BCA^TM Protein-Assay (Thermo Fisher Scientific). NPP activity was defined as 1 μmol substrate hydrolyzed per μg protein per hour.
Serum creatinine was assessed with Jaffe’s kinetic methods (IDMS standardized), serum calcium was measured by absorption spectrophotometry, serum Pi was measured with UV methods on Cobas 8000 (RocheDiagnostics, Mannheim, Germany). 25-hydroxy vitamin D (25-OH vit D) was assessed by immunologic luminescence (Diasorin^®, Fallugia, Italy). Plasma ALP activity was assessed by absorbance at 450 nm of paranitrophenol at alkaline pH. Bone ALP isoform was measured using an ¹²⁵I-labeled sandwich radioimmunoassay (Immunotech, Beckman-Coulter Society, Marseille, France). Bio-intact parathyroid hormone (PTH) was measured by luminescence immunoassay (Advia Centaur Intact PTH, Siemens healthcare, Tarrytown, United States). Osteocalcin was assessed by immunoassay (ECLIA, RocheDiagnostics, Mannheim, Germany) and luminescence was determined with an automated device (Liaison XL, Diasorin^®, Fallugia, Italy).