Appropriate methods for co-staining require that isotypes are distinct and secondary antibodies do not cross-react unless fluorophore-conjugated primary antibodies are used

Appropriate methods for co-staining require that isotypes are distinct and secondary antibodies do not cross-react unless fluorophore-conjugated primary antibodies are used. strategy is technically simple to implement and cost-effective. IL17B antibody Characterization of cardiomyocytes derived ORM-10103 from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. ORM-10103 Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described. model of ORM-10103 very early human cardiac developmental processes, providing insight into stages not otherwise accessible for mechanistic studies. This model system provides unique opportunities to study the molecular pathways that control cardiac lineage commitment and cell fate specification. In recent years, the ability to efficiently generate cardiomyogenic cells from hPSCs has greatly improved1-15. However, among protocols there is cell line variation with respect to the efficiency in generating cardiomyogenic cells and timing at which the cells express chamber-specific markers (differentiation, making it difficult to compare efficiency of cardiomyogenesis among protocols1,2,9,11. For that reason, monoclonal antibodies are used when available for all flow cytometry analyses. Going forward, it is expected that standardization of these staining protocols, especially with regards to quantitation, should better permit comparison among differentiation strategies. The choice of markers, and their corresponding antibodies, used to assess purity of differentiation arise from the fact that these gene products may not be restricted to a specific chamber throughout cardiac development, from heart tube through adult. In the rodent looped heart, MLC2a mRNA is predominant in the atrial/inflow tract area and MLC2v mRNA is predominant in the ventricular/outflow tract regions. In the looped heart, co-expression of MLC2a and MLC2v mRNAs are observed in the inflow tract, atrioventricular canal, and the outflow tract19,20. By 3 days after birth, MLC2v mRNA is restricted to the ventricle and by 10 days after birth, MLC2a is restricted to the atria in the neonatal rat heart19. Therefore, interpretation of data regarding cardiomyogenesis efficiency and subtype identity must not only consider the presence and quantity of reference marker levels, but must consider the developmental stage(s) to which the timepoints of differentiation that are analyzed correspond. This is especially important considering that the maturation stage of cardiomyogenic cells generated by differentiation of hPSCs resembles most closely those of embryonic/fetal development21-25. Thus, relying on a markers spatial expression in the postnatal heart may not be appropriate for the assessment of hPSC-derived cells, at least in some cases. In an effort to facilitate the development of more specific criteria for defining cardiomyocyte identity as it is restricted to cardiac muscle throughout embryogenesis in chick and zebrafish15,20 and is absent in human fetal skeletal muscle26. While TNNI1 is present in human fetal heart, TNNI3 is the only TNNI isoform present in normal adult heart27,28. Regarding cardiomyocyte subtype identity, IRX429-31 is an informative marker of cells with a ventricular fate. At the protein level, IRX4 has recently been shown to be restricted to the ventricle from linear heart tube through neonatal stages in the mouse32. Accordingly, optimized staining protocols for the analysis of TNNI3 and IRX4 by flow cytometry are described. To our knowledge, this is the first description of a method for efficient antibody-based staining and analysis of IRX4 levels in human cardiomyocytes by flow cytometry. Protocol 1. Solution and Media Preparation hESC Qualified Matrix Coating Stock Solution Slowly thaw hESC qualified matrix (5 ml) on ice at 4 oC overnight. Dispense.