Computational protocol: Pluripotent Stem Cell‐Derived Hematopoietic Progenitors Are Unable to Downregulate Key Epithelial‐Mesenchymal Transition‐Associated miRNAs

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Protocol publication

[…] Cells were sorted on a FACSARIA (BD Biosciences, San Jose, CA, USA) with markers CD34, CD45, CD41a, CD235a, (BD Biosciences), and CD43 (Life Technologies). Cells were analyzed after miRNA inhibition on a LSRII (BD Biosciences) flow cytometry machine using the same markers. Results were analyzed using BD FACSDIVA software. At least 10,000 events were acquired for each experiment. [...] Six thousand unsorted cells were taken from differentiation culture and transferred to a 3‐ml aliquot of MethoCult media and plated into two 10‐mm cell culture dishes scored with 2‐mm grids. These were then incubated for 14 days. Colonies in each dish were counted and scored based on their morphology. Inhibitions were compared with control data using univariate one‐way analysis of covariance with effect of passage number on numbers of hematopoietic progenitors as the covariate (see Supporting Information Figs. 1, 2). p values are calculated as Sidak corrected bootstrapped significance. Analysis was performed using SPSS statistics software. Figures show estimated marginal means. […]

Pipeline specifications

Software tools BD FACSDiva, SPSS
Applications Miscellaneous, Flow cytometry