Purified Rat Anti-Mouse CD106

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Embryonic heart cell suspensions were stained on ice in PBS containing 5% FCS using the following pre-titrated antibodies	rat anti VCAM-1 (10 µg/ml, BD Biosciences)	Cell sorting was performed on a FACSAria (BD Biosciences) with 100 µM nozzle. Doublet cells were discriminated as previously described [17].

Upstream tips
Embryonic heart cell suspensions were stained on ice in PBS containing 5% FCS using the following pre-titrated antibodies
Protocol tips
rat anti VCAM-1 (10 µg/ml, BD Biosciences)
Downstream tips
Cell sorting was performed on a FACSAria (BD Biosciences) with 100 µM nozzle. Doublet cells were discriminated as previously described [17].

Publication protocol

Embryonic heart cell suspensions were stained on ice in PBS containing 5% FCS using the following pre-titrated antibodies: rat anti VCAM-1 (10 µg/ml, BD Biosciences), mouse anti-rat PE (2 µg/ml, BD Biosciences), rat anti PECAM-1 -APC (0.5 µg/ml, BD Biosciences), or isotype controls rat-IgG2a and rat-IgG2a -APC at corresponding concentrations (BD Biosciences). Positive sorting gates for the VCAM-1 high positive and PECAM-1 negative cells were set according to unstained controls, isotype controls and single staining controls. To exclude non-viable cardiomyocytes, cells were also stained with the DNA binding dye To-pro-1 (Invitrogen) or 7-AAD (Sigma-Aldrich). Cell sorting was performed on a FACSAria (BD Biosciences) with 100 µM nozzle. Doublet cells were discriminated as previously described [17]. Viability of cardiomyocytes was assessed during every round of sorting as well as after sorting (aliquots of 100–200 of the sorted cells). In a thorough viability analysis, 50.000 sorted cells were stained with To-pro-1 and re-analyzed by FACS. The purity of cells gated for VCAM-1+ PECAM-1− was also assesses after each isolation. Small aliquots of cells (100–200 of the sorted cells) were re-analyzed using the same settings for gating. The fraction of cells still within the positive sorting served as a quality control of cell purity. For cardiomyocyte purity analysis, FACS isolated cells were fixed with 2% PFA, permeabilized and stained in Perm/Wash solution (BD Biosciences) with mouse anti cTropT-FITC antibodies (9 µg/ml, Hytest Ltd), or isotype control antibodies at corresponding concentration (mouse IgG2b-FITC, BD Biosciences). Neonatal heart cell preparations served as positive control.

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