Crude collagenase

A 2×2.5-cm piece of depilated back skin was minced and digested in 7 ml of RPMI 1640 containing 10% FBS with 2 mg/ml crude collagenase (Sigma-Aldrich), 1.5 mg/ml hyaluronidase (Sigma-Aldrich), and 0.03 mg/ml DNase I (Roche Applied Science) at 37 ^oC for 90 min. The samples were passed through a 70-μm Falcon cell strainer (BD Biosciences) to obtain single-cell suspensions. After centrifugation at 1500 rpm for 5 min, the cell pellet was resuspended in a 70% Percoll solution (GE Healthcare) and then overlaid with a 37% Percoll solution, followed by centrifugation at 1800 rpm for 20 min. The cells were aspirated from the Percoll interface and passed through a 70-μm Falcon cell strainer. The harvested cells were washed with ice-cold PBS and were used for flow cytometric analysis.

MV were isolated from calcifying VSMC obtained from three CKD rats by collagenase digestion as previously described [4 (link)]. Calcifying VSMC were incubated with crude collagenase (500 U/ml, type IA, Sigma) in a solution of 0.25 M sucrose, 0.12 M NaCl, 0.01 M KCL and 0.02 M Tris buffer, pH 7.45, at 37°C for 3 hrs. The digests were centrifuged at 800 g and 30,000 g to remove cell debris and microsomes, respectively. The supernatant was centrifuged at 250,000 g to pellet the MV followed by resuspension in TBS (pH 7.6) with 0.25 M sucrose. The MV amount was determined by protein concentration (Bio-Rad). We used 3 different CKD rats to isolate 3 different VSMC cultures. Each of these three cultures was used to isolated MV. We then ran separate arrays on each of the VSMC-MV pairs.

A 2 × 2.5-cm piece of depilated back skin was minced and then digested in 7 ml of RPMI 1640 medium containing 10% FBS and 2 mg/ml crude collagenase (Sigma-Aldrich, St. Louis, MO, USA), 1.5 mg/ml hyaluronidase (Sigma-Aldrich), and 0.03 mg/ml DNase I (Roche Applied Science, Indianapolis, IN, USA) at 37 °C for 90 minutes [23 (link)]. Samples were passed through a 70-μm Falcon cell strainer (Fisher Scientific/BD Biosciences, Pittsburgh, PA, USA) to obtain single-cell suspensions. After centrifugation at 1500 rpm for 5 minutes, the cell pellet was resuspended in a 70% Percoll solution (GE Healthcare Life Sciences) and then overlaid with a 37% Percoll solution, followed by centrifugation at 1800 rpm for 20 minutes. The cells were aspirated from the Percoll interface and passed through a 70-μm Falcon cell strainer. The harvested cells were washed with ice-cold PBS and used for flow cytometric analysis.