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Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit

Catalog #: SC021
4 Citations Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
SC021
Verification of Human Embryonic Stem Cell Pluripotency.
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Citations (4)
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Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit Summary

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.

 

Specifications

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.
Applications
Immunocytochemistry
Species
Human

Product Datasheets

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Scientific Data

Immunocytochemistry Verification of Human Embryonic Stem Cell Pluripotency. View Larger

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.

Immunocytochemistry Analysis of Human Stem Cell Pluripotency.  View Larger

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.

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
SC021 Protocol Graphic Summary
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)

Materials

  • Human pluripotent stem cells
  • Coverslips (sterilized)
  • Cell culture plate (24-well)

Equipment

  • 37 °C, 5% CO2 incubator
  • Centrifuge
  • Hemocytometer
  • Inverted microscope
  • Fluorescence microscope
 

 

Procedure Overview

Coat coverslips with a feeder cell layer or a defined matrix.

Culture cells to 100% confluency.

Plate ES or iPS cells.

Culture to desired confluency/age.

Replace the medium with Adipogenic Differentiation Medium to induce adipogenesis.

Fix stem cells with 4% paraformaldehyde.

ICC detection of FABP4.

Block with blocking solution.

Plate 4.2 x 103 MSCs/cm 2 in StemXVivo Osteogenic/Adipogenic Base Media.

Incubate with fluorochrome-conjugated primary antibodies.

Wash with wash buffer.

Replace the medium with Osteogenic Differentiation Medium to induce osteogenesis.

Incubate with nuclear counterstain.

ICC detection of Osteocalcin.

Mount the coverslip.

Visualize using a fluorescence microscope and appropriate filter sets.

Transfer 2.5 x 104 MSCs to a 15 mL conical tube.

Citations for Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

4 Citations: Showing 1 - 4
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  1. Joint epigenome profiling reveals cell-type-specific gene regulatory programmes in human cortical organoids
    Authors: Noack, F;Vangelisti, S;Ditzer, N;Chong, F;Albert, M;Bonev, B;
    Nature cell biology  2023-11-23
  2. Generation of a human induced pluripotent stem cell line derived from a patient with dilated cardiomyopathy carrying LMNA nonsense mutation
    Authors: Y Shimoda, N Murakoshi, H Mori, D Xu, K Tajiri, Y Hemmi, I Sato, M Noguchi, Y Nakamura, Y Hayashi, M Ieda
    Stem Cell Research, 2022-04-27;62(0):102793.  2022-04-27
  3. Direct reprogramming of epithelial cell rests of malassez into mesenchymal-like cells by epigenetic agents
    Authors: K Yoshida, O Uehara, Y Kurashige, D Paudel, A Onishi, P Neopane, D Hiraki, T Morikawa, F Harada, R Takai, J Sato, M Saitoh, Y Abiko
    Scientific Reports, 2021-01-20;11(1):1852.  2021-01-20
  4. Organoid single-cell genomic atlas uncovers human-specific features of brain development
    Authors: S Kanton, MJ Boyle, Z He, M Santel, A Weigert, F Sanchís-Ca, P Guijarro, L Sidow, JS Fleck, D Han, Z Qian, M Heide, WB Huttner, P Khaitovich, S Pääbo, B Treutlein, JG Camp
    Nature, 2019-10-16;574(7778):418-422.  2019-10-16

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