Analysis of the trilineage differentiation potential of human pluripotent stem cells (PSCs) using flow cytometry

MBTP 22 = Miltenyi Biotec-tested panel 22

This application protocol describes the quantitative analysis of the trilineage differentiation potential of human pluripotent stem cells (PSCs) using flow cytometry. To demonstrate this state-of-the-art panel for pluripotent stem cell differentiation, PSCs were differentiated using the StemMACS™ Trilineage Differentiation Kit. The StemMACS™ Trilineage Differentiation Kit enables the directed and reproducible differentiation of stem cells into ectodermal, mesodermal, and endodermal lineages ready for downstream applications, such as flow cytometric analysis.

Protocol

Antibody panel

SpecificityClonePurposeFluorochromeDetection filter (nm) (laser)
CD144 (VE-Cadherin)REA199MesodermFITC525/50 (blue)
CD140bREA363MesodermAPC655-730 (red)
CD184 (CXCR4)REA649EndodermAPC655-730 (red)
Sox17REA701EndodermVio® B515525/50 (blue)
PAX-6REA507EctodermAPC655-730 (red)
Sox2REA320EctodermFITC525/50 (blue)
Propidium Iodide Solution-Viability-655-730 (blue)

Gating strategy

Gating strategy showing the analysis of the trilineage differentiation potential of human PSCs. Human PSCs were stained using the previously described panel to analyze their trilineage differentiation potential. Exclusion of debris using SSC-A/FSC-A gating was initially performed on meso-, ecto-, and endoderm samples, respectively (A, B, C). Propidium iodide was added to mesoderm samples shortly before flow cytometric analysis for exclusion of dead cells (D). No dead cell exclusion was done for endo- and ectoderm samples, since these cells were fixed. For mesoderm samples CD140b+ and CD144+ cells were gated (E), for ectoderm Sox2+ PAX-6+ cells (F), and for endoderm Sox17+ CD184 (CXCR4)+ cells (G).

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Materials

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