Q: In Figure 1 on the Datasheet for Catalog # SC022, why is the staining produced by the antibody conjugated to NL637 dye green in one image and red in another image?

A: NL637 is assigned a different color (green) than its natural color in one of the the images in order to obtain good contrast with the co-staining done for another marker in the same sample. Red is the natural color for NL637.

Human Three Germ Layer 3-Color Immunocytochemistry Kit

R&D Systems | Catalog # SC022

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Product Documents

Contains six fluorochrome-conjugated anti-human antibodies: Ectoderm (SOX1, Otx-2); Mesoderm (Brachyury, HAND1); and Endoderm (GATA-4, SOX17)

Discontinued Product

SC022 has been discontinued. View all Embryonic Stem Cells products.

Product Summary
Scientific Data
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Species

Human

Product Summary for Human Three Germ Layer 3-Color Immunocytochemistry Kit

Kit Summary

To analyze germ layer cells differentiated from human pluripotent stem cells.

Key Benefits

Verifies human stem cell pluripotency
Labels cells of the ectoderm, mesoderm, and endoderm
Can be used for simultaneous 3-color staining

Why Use Molecular Markers to Verify Human Stem Cell Pluripotency?

Research studies using embryonic stem (ES) or induced pluripotent stem (iPS) 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 is an important time saving step. Pluripotency status can be evaluated by the presence of stem cell markers and/or the ability of stem cells to differentiate into cells of the three germ layers. To determine if a cell is truly a pluripotent stem cell, it is important to verify its ability to differentiate into each of the three germ layers.

3-Color ICC using germ layer markers:

Evaluates the pluripotency status of the starting stem cell population.
Provides increased confidence in pluripotent status through the use of multiple markers for each germ layer.
Ensures efficient use of time and reagents by utilizing single-step staining.
Can be performed in randomly differentiated samples to simultaneously detect one marker from each germ layer.

Kit Contents

This kit contains the following fluorochrome-conjugated antibodies to analyze human pluripotent stem cells differentiated into cells of each germ layer:

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

Each antibody is supplied as a 10X solution; enough for 25 assays when used in a 50 µL staining volume per assay.

Stability and Storage

Reagents are stable for 12 months from date of receipt when stored in the dark at 2^° C to 8^°.

Scientific Data Images for Human Three Germ Layer 3-Color Immunocytochemistry Kit

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 (NL) fluorochrome 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.

Random Differentiation within a BG01V Human Embryonic Stem Cell-derived Embryoid Body.

BG01V human embryonic stem cell-derived embryoid bodies were grown on plates coated with Cultrex^®Stem Cell Qualified Basement Membrane Extract (Catalog # 3434-005-02). Random differentiation was detected by simultaneous 3-color staining using three fluorochrome-conjugated antibodies provided in the Human Three Germ Layer 3-Color Immunocytochemistry Kit (Catalog # SC022). Ectoderm derivatives were detected with NorthernLights (NL) fluorochrome NL557-conjugated Otx2 (red), mesoderm differentiated cells with NL637-conjugated HAND1 (white), and ectoderm derivatives with NL493-conjugated GATA-4 (green). The nuclei were counterstained with DAPI (blue).

Formulation, Preparation, and Storage

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: Embryonic Stem Cells

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.

R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.

Additional Embryonic Stem Cells Products

Product Documents for Human Three Germ Layer 3-Color Immunocytochemistry Kit

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Product Specific Notices for Human Three Germ Layer 3-Color Immunocytochemistry Kit

For research use only

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