FlowCellect Human ESC Nuclear Marker Characterization Kit

FlowCellect Human ESC Nuclear Marker Characterization Kit

FlowCellect

Millipore’s The FlowCellect™ Human Embryonic Stem Cell Characterization kit is designed as an easier and quicker way to track surface marker expression of hOCT4, SSEA4 and SSEA1. The kit will also help to determine the percentage of undifferentiated human embryonic stem cells in culture by determining the percentage of cells that express both hOCT4 and SSEA4 and not SSEA1. This quick test will allow researchers to determine the quality of their cells in culture as well as see changes in marker expression during a differentiation protocol.
Sufficient reagents are provided for 25 3-color samples and 5 isotype control tests. The kit includes all optimized fluorescently labeled antibodies and buffers necessary for analysis. To verify antibody specificity, all antibodies were tested against the ENSTEM-A cell line that does not express hOCT4, SSEA4 or SSEA1. A cell strainer is also included to ensure samples are devoid of large cell clumps that can alter cell analysis. Detailed assay instructions are also included to assist in analysis and to ensure the correct cell concentration is obtained for flow cytometric analysis.

Human embryonic stem cells (hESCs) are unique cells in that they can be maintained in vitro as pluripotent cells and can be induced to differentiate into many cell types by manipulating culture conditions. Therefore, hESCs have the potential of providing an unlimited supply of cells for cell-based therapy as well as providing a resource to study basic human developmental biology. Subsequently, much attention has been focused on directing hESC differentiation along specific developmental lineages [1-4].

Currently, the most utilized method to test for the undifferentiated state of hESCs is to analyze the expression of the proteins OCT4, NANOG, Alkaline phosphatase, SSEA3, SSEA4, TRA-1-60, and TRA-1-81 [5]. The transcription factor, OCT4 is essential for the formation and maintenance of pluripotent embryonic stem cells (6,7). High levels of OCT4 expression is seen in undifferentiated hESCs and is downregulated upon differentiation (8), thus making OCT4 an important marker to study hESCs. The stage specific embryonic antigens or SSEAs (SSEA1, 3 and 4) are also good markers of undifferentiated hESCs (9). The antibodies for SSEAs recognize epitopes on related glycosphingolipids or GSLs (10). The SSEA4 antibody recognizes the terminal sialic acid on GSL-7 (10). Upon differentiation, SSEA4 expression levels decrease and SSEA1 expression increases on human embryonic stem cells over time when treated with Retinoic acid (11).

The FlowCellect™ Human Embryonic Stem Cell Characterization kit uses an antibody-based detection method, utilizing fluorescently conjugated antibodies to detect hOCT4, SSEA4 and SSEA1. The FlowCellect™ Human Embryonic Stem Cell Characterization kit is designed specifically for flow cytometers with a single blue (488 nm) laser light source. The FlowCellect™ Human Embryonic Stem Cell Characterization kit is not intended for use in microscopy applications including ICC, IHC and HTS, western blot or any other fluorometric analysis platforms.

The kit is optimized for use on the Guava EasyCyte™ Plus cytometer that requires cell concentrations to be less than 500 cells per microliter. The kit does not include culture media or other materials that should be readily available in a common laboratory setting that routinely performs experiments of the type described here.

• Flow Cytometry
• in vitro Toxicology Assays

Human

• Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
• Alexa Fluor® is a registered trademark of Molecular Probes, Inc.

Lot to lot in-process testing on all kit components

Stem Cell Kits

Human Embryonic Stem Cells

Stem Cell Research

Stem Cell

Characterization

• 1. 10X Wash buffer: (Part No. CS202123) 1 bottle containing 13mL
• 2. Fixation buffer: (Part No. CS202122) 1 bottle containing 13mL
• 3. 5X Assay buffer: (Part No. CS202124) 1 bottle containing 55mL
• 4. 1X Permeabilization Buffer: (Part No. CS202125) 1 bottle containing 13mL
• 5. 20X IgG-Alexa 488 isotype control: (Part No. CS202402) 1 vial containing 75uL
• 6. 20X IgG3-PE isotype control: (Part No. CS202405) 1 vial containing 75uL
• 7. 20X IgM-PE/CY5 isotype control: (Part No. CS202397) 1 vial containing 75uL
• 8. 20X hOCT4-Alexa 488: (Part No. CS202403) 1 vial containing 150uL
• 9. 20X SSEA4-PE: (Part No. CS202399) 1 vial containing 150uL
• 10. 20X SSEA1-PE/CY5: (Part No. CS202400) 1 vial containing 150uL
• 11. Steri-flip cell strainer (60mm): (Catalog # SCNY00060)

4-8°C; 6 Months

Mouse

Fluorescent

• POU5F1
• SSEA-4
• FUT4

Flow Cellect

ExpressPro

Monoclonal Antibody

Research