Pe conjugated mouse igg1

FACS analysis of whole blood samples was performed at baseline, 3d post MI and at 1 month FUP in order to address the kinetics of mobilized CD34+ cells
in vivo. EDTA-treated venous blood samples (100μl) were labelled with PE-DY647-conjugated CD34+ antibody (monoclonal antibody; host/isotype: mouse/IgG1; cat# MA1-19770; Thermo Fisher Scientific, Waltham, MA, USA) or the corresponding isotype control (PE-conjugated mouse IgG1; cat# MA1-10415; Thermo Fisher Scientific, Waltham, MA) for 20min at room temperature. Anti-human CD34+ antibody was utilized due to lack of commercially available porcine-specific CD34+ marker (dilution: 5μl antibody/100μl whole porcine blood). Subsequently, erythrocyte cell lysis was performed, according to the manufacturer’s protocol, using Dako-Uti Lyse
^TM (Dako, Agilent Technologies, Santa Clara, CA, USA) following fixation with PBS containing 1% paraformaldehyde. FACS analysis was performed on CyFlow
^® ML/space flow cytometer (Sysmex Partec, Görlitz, Germany) with acquired 100.000 events within the gated region of mononuclear cells of forward versus side scatter. Absolute counts of CD34+ cells were obtained by multiplying the ratio of the CD34+ cells obtained in the flow cytometry analysis and absolute count of leucocytes per 1μl of blood.

The following mouse monoclonal antibodies (mAbs) were used: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnology, Heidelberg, Germany) and anti-transient receptor potential cation channel A1 (TRPA1) (Santa Cruz Biotechnology), anti-class III-beta-tubulin (β_III-tubulin) (Millipore, Billerica, MA, USA), anti-Aml1 (Cell Signaling Technology, Danvers, MA, USA). The following polyclonal Abs were used: rabbit anti-glial fibrillary acidic protein (GFAP) and rabbit anti-transient receptor potential vanilloid 1 (TRPV1) (ThermoFisher Scientific, Rodano, Italy). The following secondary Abs were used: horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG and HRP-conjugated donkey anti-rabbit IgG (Cell Signaling Technology), anti-mouse Alexa Fluor-488 (ThermoFisher Scientific), anti-rabbit Alexa Fluor-594 (ThermoFisher Scientific), PE-conjugated anti-rabbit (Santa Cruz Biotechnology), PE-conjugated anti-mouse (Santa Cruz Biotechnology). The following isotypes were used: PE-conjugated mouse IgG1 and PE-conjugated rabbit IgG1 (ThermoFisher Scientific).

The 3rd–5th passages of MSCs were harvested by trypsinization and incubated with 10 μL of the following mouse anti-human antibodies: anti-CD34-FITC (BD Pharmingen, USA), anti-CD45-PE (BD Pharmingen, USA), anti-CD90-FITC (AbD Serotec, USA), anti-CD73-PE (BD Pharmingen, USA), and anti-CD105-PE (Miltenyi Biotec, Germany) for 30 minutes at 4°C in the dark. After incubation, cells were washed twice with PBS and fixed with 300 μL 1% (v/v) paraformaldehyde. The expression profiles of cell surface markers were then determined by FACSCalibur flow cytometry (Becton Dickinson, USA) using CellQuest software. Cells labeled with FITC-conjugated mouse IgG1 (eBioscience, USA) and PE-conjugated mouse IgG1 (eBioscience, USA) served as negative controls.