Cytexpert acquisition and analysis software

Phenotypical characterization of hAFSCs was performed between passages 2 and 4, as previously described (Di Baldassarre et al., 2018b (link)). Briefly, cells were treated with the FIX & PERM^® Kit (Thermo Fisher Scientific) and then incubated for 1 h at RT with human anti-CD90-Alexa fluor 488-conjugated, CD34-Alexa fluor 488-conjugated, CD45-Alexa fluor 488-conjugated, SSEA4-Alexa fluor conjugated, OCT4-Alexa fluor 488-conjugated, Tra-1–60-Alexa fluor 488-conjugated, C-KIT-PE-conjugated, CD105-FITC-conjugated, NANOG-Alexa fluor 647-conjugated, SOX2-Alexa fluor 488-conjugated (All from Becton Dickinson, Franklin Lakes, NJ, United States) diluted 1:50–1:100 according to the manufacturer’s instructions. Cells incubated with isotypes (all from Becton Dickinson) were used as negative controls. Cytometric analysis was performed with a Cytoflex cytometer (Beckman Coulter Pasadenia, CA, United States), and data were analyzed with CytExpert Acquisition and Analysis Software (Beckman Coulter).

We lysed 100-μl citrated whole blood samples at room temperature for 10 min using Lysing Solution 10X Concentrate (349202; BD Biosciences). Lysed blood was then centrifuged at 4 °C and 600 × g for 6 min, and the pellet re-suspended in HEPES buffered saline (Sigma-Aldrich). Cultured cells were also re-suspended in HEPES buffered saline, to a final concentration of 10⁵ cells/100 μl. Rat Anti-Human EPCR monoclonal antibodies and isotype controls were added as appropriate at a final concentration of 0.125 μg/100 μl and incubated at room temperature for 20 min in the dark. Samples were diluted in 0.5 ml ice-cold HEPES buffered saline prior to flow cytometric analysis using either a Cytomics FC500 with Cytomics CXP software v2.2 or a CytoFLEX S Flow Cytometer with CytExpert Acquisition and Analysis software v2.3 (Beckman Coulter). CD14⁺ Monocytes and CD16⁺ neutrophils from blood lysates were gated using forward and side light scatter, enabling discrimination by cell size and granularity, respectively. The gating strategy was validated using mouse anti-human CD14 and CD16 antibodies (Supplementary Fig. 7) added to blood lysates at a final concentration of 0.125 μg/100 μl. Results were recorded as median fluorescence intensity.

For flow cytometry, the cells were treated with the FIX & PERM^® Kit (Thermo Fisher Scientific, MA, USA) and then, incubated for 1 h at RT with anti-Oct-4 Alexa Fluor 488 conjugated (Cell Signaling, Danvers, MA, USA) 1:50, anti-Nanog Alexa Fluor 647 conjugated (Becton Dickinson, Franklin Lakes, NJ, USA) 1:20, anti-c-Kit PE conjugated (Becton Dickinson, NJ, USA) 1:5, anti-Sox2 Alexa Fluor 488 conjugated (Becton Dickinson, NJ, USA) 1:5, anti-OVOL-1 (Abcam, Cambridge, UK) 1 µg/mL, anti-KLF4 (Thermo Fisher Scientific, Waltham, MA, USA) 1:1000, anti-ESG1 (Abcam, Cambridge, UK) 1:50, anti-REX1 (Abcam, Cambridge, UK) 1:200 or appropriate isotype controls (all from Becton Dickinson, NJ, USA), followed by appropriate secondary antibody Alexa Fluor 488 if necessary (1:100, Invitrogen, Carlsbad, CA, USA). Cytometric analyses were performed with a Cytoflex cytometer (Beckman Coulter, CA, USA), and the data were analyzed with CytExpert Acquisition and Analysis Software (Beckman Coulter, CA, USA).

The exosomal markers (CD63 and CD9) on the surfaces of SEVs were assayed using an Exo-FACS kit (HansaBioMed Life Science, Tallinn, Estonia), according to the protocol. SEVs immobilized on the surface of the latex beads were labeled with monoclonal antibodies to tetraspanins CD63 or CD9, conjugated with FITC or PerCP-Cy5.5, respectively. The flow cytometry data were obtained on a CytoFLEX analyzer (Beckman Coulter, Brea, CA, USA) equipped for multi-parametric and multicolor analysis, including an argon laser set at 488 nm for measurements of forward light scatter (FSC) and orthogonal scatter (SSC). The complexes assembled without SEVs were used as negative controls. The data were analyzed with CytExpert Acquisition and Analysis Software (Beckman Coulter, Brea, CA, USA).

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).