Isotypes

Circulating endothelium-, monocyte- and platelet-derived microparticles were measured as previously reported (21 (link),22 (link)). In brief, blood samples were collected and centrifuged (160g; 20-22°C; 10 min) to obtain platelet-rich plasma (PRP). The PRP was centrifuged (1500g; 20-22°C; 6 min) to obtain platelet-poor plasma (PPP). Furthermore, PPP (50 µL) was labeled (20 min; room temperature) with CD51FITC, CD42FITC and CD31 PE, and CD14FITC (BD Biosciences) for the identification of endothelial, platelet, and monocytic microparticles, respectively. Isotypes (BD Biosciences) were used as controls. Microparticles were quantified per microliter of PPP injected into the cytometer, according to the standard protocol. TruCOUNT (BD Biosciences) tubes containing a known number of beads were used to quantify the number of microparticles per microliter of PPP.

For flow cytometry and imaging flow cytometry, cells were treated with the FIX and PERM^® Kit (Thermo Fischer Scientific, Waltham, MA, USA) and incubated for 1 h at RT with anti-α-myosin heavy chain (α-MHC; 1:100; Abcam, Cambridge, UK), anti-cardiac troponin T (cTnT, 1:100; Abcam, Cambridge, UK), anti-α-sarcomeric actin (α-SA; 1:100; Abcam, Cambridge, UK), anti-sarcolemmal Ca²⁺ channel (CACNA1C, 1:100; Abcam, Cambridge, UK), and anti-sarcoendoplasmic reticulum Ca²⁺ ATPase protein (Serca2, 1:100; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), followed by incubation with the appropriate secondary antibody conjugated with Alexa Fluor 488, PE-Alexa Fluor 647, or PE-Cy7 (1:200; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), as previously reported [53 (link)]. Cells incubated with isotypes (all from BD) were used as negative controls [54 (link)].
Cytometric analysis was performed with a Cytoflex cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) or CytExpert Acquisition and Analysis Software (Beckman Coulter, Brea, CA, USA).