Name: Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit
Brand: R&D Systems
SKU: SC021

Kit Summary

To verify pluripotency in human stem cells using single-step ICC.

Key Benefits

Defines pluripotency in the starting stem cell population
Reduces experimental variation
Utilizes single-step, fluorescent ICC staining of pluripotency markers

Why Verify the Pluripotency of a Human Stem Cell Population?

Maintaining and expanding healthy, undifferentiated stem cells in culture can be challenging if spontaneous differentiation occurs.

In addition, research studies using embryonic or induced pluripotent stem cells most often require weeks of cell culture before an experimental hypothesis can be tested.

Verifying stem cell pluripotency using 3-color ICC:

Defines the starting stem cell population.
Increases confidence in pluripotent status by combining 3 different fluorochrome-conjugated antibodies for simultaneous staining.
Reduces variability within an experiment and between studies.
Allows researchers to expand and study high quality, undifferentiated stem cell starting populations.
Single-step staining ensures efficient use of time and reagents.

Kit Components

This kit contains the following fluorochrome-conjugated antibodies to verify human stem cell pluripotency:

NL557-conjugated Goat Anti-Human SOX2
NL637-conjugated Goat Anti-Human Oct-3/4
NL493-conjugated Goat Anti-Human Nanog

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

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.

Product Specifications for Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit

Species	Human
Applications	Immunocytochemistry/ Immunofluorescence
Source	N/A

Scientific Data Examples for Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit

	Verification of Human Embryonic Stem Cell Pluripotency. BG01V human embryonic stem cells grown on Irradiated Mouse Embryonic Fibroblasts (Catalog # PSC001) were simultaneously stained with the following antibodies supplied in the Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit (Catalog # SC021): NL637-conjugated Oct-3/4, NL493-conjugated Nanog, and NL557-conjugated SOX2. The nuclei were counterstained with DAPI. The uniform expression of three stem cell markers increases confidence in the pluripotent status of this stem cell population. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
	Analysis of Human Stem Cell Pluripotency. iPS2 human induced pluripotent stem cells were grown on StemXVivo^®Culture Matrix (Catalog # CCM013) in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog # AR005). The cells were simultaneously stained with NL637-conjugated Oct-3/4 (pseudocolored green) and NL557-conjugated SOX2 (red) antibodies, supplied in the Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit (Catalog # SC021). Co-staining reveals that some cells are negative for SOX2, suggesting that the population of Oct-3/4⁺cells is not uniform and some of the cells may be initiating differentiation. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Preparation & Storage

Shipping Conditions	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.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, human stem cell pluripotency can be verified via 3-Color ICC using the following protocol:

Pluripotent stem cells are cultured on coated coverslips
Fluorochrome-conjugated primary antibodies are added to fixed stem cells
Pluripotency markers are visualized using a fluorescence microscope

View Graphic Procedure

Reagents Provided

Reagents supplied in the Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit (Catalog # SC021):

NL557-conjugated Goat Anti-Human SOX2
NL637-conjugated Goat Anti-Human Oct-3/4
NL493-conjugated Goat Anti-Human Nanog

Other Supplies Required

Reagents

Appropriate stem cell culture substrate (e.g., StemXVivo^® Culture Matrix (Catalog # CCM013), iMEFs (Catalog # PSC001), etc.)
Cell culture medium
4% paraformaldehyde in PBS
1% BSA in PBS
0.3% Triton^™ X-100, 1% BSA, 10% normal donkey serum in PBS
Mounting medium (Catalog # CTS011 or equivalent)