Human Three Germ Layer 3-Color Immunocytochemistry Kit

For the verification of stem cell pluripotency.

Kit Summary

• Six fluorochrome-conjugated antibodies to verify the ability of pluripotent stem cells to differentiate into cells of the ectoderm, mesoderm, and endoderm.
• Two antibodies are provided for each germ layer.

  • Ectoderm: Otx2 & SOX1
    Mesoderm: Brachyury & HAND1
    Endoderm: GATA-4 & SOX17

• Can be used for simultaneous 3-color staining.

Why is it Important to Verify Stem Cell Pluripotency?

Pluripotent stem cells are characterized by their ability to self-renew and to differentiate into cells of the three germ layers. Research studies using embryonic or induced pluripotent stem cells most often require weeks of cell culture before an experimental hypothesis can be tested. Thus, the ability to verify pluripotency at an early stage in the experimental process:

• Defines the starting stem cell population.
• Reduces variability within an experiment and between studies.
• Ensures efficient use of time and reagents.

Kit Contents

R&D Systems™ Human Three Germ Layer 3-Color Immunocytochemistry Kit (Catalog # SC022) contains six fluorochrome-conjugated antibodies to detect germ layer-specific markers. Assessing the expression of multiple markers for each lineage provides additional confidence in determining the pluripotent status of the starting stem cell population. In addition, these antibodies are compatible with use for 3-color staining in randomly differentiated samples to simultaneously detect one marker from each germ layer.

Protocol for Fluorescent
ICC Staining

Ectoderm

• Anti-Human Otx2 NL557-Conjugated Goat IgG
• Anti-Human SOX1 NL493-Conjugated Goat IgG

Mesoderm

• Anti-Human Brachyury NL557-Conjugated Goat IgG
• Anti-Human HAND1 NL637-Conjugated Goat IgG

Endoderm

• Anti-Human GATA-4 NL493-Conjugated Goat IgG
• Anti-Human SOX17 NL637-Conjugated Goat IgG

Typical Data

Verification of Germ Layer Markers in Differentiated Pluripotent Stem Cells. iPS2 human induced pluripotent stem cells were differentiated into each of the three germ layers using the base media and differentiation supplements provided in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). Germ layer differentiation was subsequently verified using the six fluorochrome-conjugated antibodies provided in the Human Three Germ Layer 3-Color Immunocytochemistry Kit (Catalog # SC022). Ectoderm differentiated cells were simultaneously stained with NorthernLights™ flourochrome (NL) NL557-conjugated Otx2 (red) and NL493-conjugated SOX1 (green). Mesoderm differentiated cells were simultaneously stained with NL557-conjugated Brachyury (red) and NL637-conjugated HAND1 (green). Endoderm differentiated cells were simultaneously stained with NL637-conjugated SOX17 (red) and NL493-conjugated GATA-4 (green). All nuclei were counterstained with DAPI (blue). Nuclear expression of each marker was detected in their respective cell lineage.

A Complete Kit for the Differentiation & Verification of Pluripotent Stem Cells
R&D Systems also offers the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027), which contains base media and differentiation supplements to verify the ability of human pluripotent stem cells to differentiate into cells of the three germ layers. To confirm the pluripotency status of the starting stem cell population, this kit also includes one antibody to detect a marker for each of the three differentiated cell types Otx2 (ectoderm), Brachyury (mesoderm), and SOX17 (endoderm).