H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit

Name: H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit
Brand: R&D Systems
SKU: FMC001

R&D Systems, part of Bio-Techne | Catalog # FMC001

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Product Datasheet / COA / SDS

Key Product Details

Summary for H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit

Kit Summary

For the verification of stem cell pluripotency using four established markers.

Why is it Important to Verify Embryonic Stem Cell Pluripotency Using Multiple Established Markers?

Identifying the sources of experimental variability is an important consideration in stem cell research where experiments are costly and time-consuming. A potential source of significant variability arises from the starting population of stem cells which can undergo phenotypic changes in culture. See Details

Changes in stem cell potency over time can give rise to large inter-assay errors and/or contradictory data. The Human/Mouse Pluripotent Stem Cell Multi-Color Flow Cytometry Kit offers users an efficient and quantitative method to verify the pluripotency of cells by flow cytometry. Data obtained using this kit can identify and minimize experimental errors introduced by variations in the starting population of cells.

The Human/Mouse Pluripotent Stem Cell Multi-Color Flow Cytometry Kit:

Efficiently verifies stem cell pluripotency using four markers of pluripotency.
Defines the starting population to reduce experimental variability.
Does not require the use of secondary antibodies.
Includes enough reagents to perform 25 assays.

Kit Components

The Human/Mouse Pluripotent Stem Cell Multi-Color Flow Cytometry Kit includes four fluorochrome-conjugated primary antibodies, isotype controls and buffers to fix, permeabilize, and wash cells. See Details

SOX2-PE (Clone 245610; mouse IgG_2A)
Oct-3/4-APC (Clone 240408; rat IgG_2B)
SSEA-1-PerCP (Clone MC-480; mouse IgM)
SSEA-4-CFS (Clone MC-813-70; mouse IgG₃)
Mouse IgM Isotype Control
Mouse IgG₃ Isotype Control
Rat IgG_2B Isotype Control
Mouse IgG_2A Isotype Control
Fixation/Permeabilization Buffer (with 1% formaldehyde, 8.9% saponin, and
Permeabilization/Wash Buffer (with 10% saponin and 0.05% sodium azide)
Includes enough reagents to perform 25 assays

Stability and Storage

Store at 2 °C to 8 °C in the dark. Use within 6 months of receipt.

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 H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit

Species	Human, Mouse
Source	N/A

Scientific Data Examples for H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit

	Verification of Human BG01V Embryonic Stem Cell Pluripotency by Multi-Color Flow Cytometry. BG01V human embryonic stem cells were stained using reagents included in the Human/Mouse Pluripotent Stem Cell Multi-Color Flow Cytometry Kit (Catalog # FMC001). Cells were simultaneously analyzed for expression of pluripotent markers including SSEA-1, SSEA-4, Oct-3/4, and SOX2 by flow cytometry. A. Flow cytometry data shows that 91.9% of BG01V human embryonic stem cells are positive for both Oct-3/4 and SSEA4 expression. B. Flow cytometry data shows that 88.5% of BG01V human embryonic stem cells are positive for SSEA-4 and negative for SSEA-1, a phenotype consistent with human embryonic stem cells. C. Flow cytometric analysis shows that BG01V human embryonic stem cells express the pluripotent marker SOX2.
	Verification of Mouse D3 Embryonic Stem Cell Pluripotency by Multi-Color Flow Cytometry. Mouse D3 embryonic stem cells were stained using reagents included in the Human/Mouse Embryonic Stem Cell Multi-Color Flow Cytometry Kit (Catalog # FMC001). Cells were analyzed for expression of pluripotent markers including SSEA-1, SSEA-4, Oct-3/4, and SOX2 by flow cytometry. A. Flow cytometric analysis shows that 91.1% of mouse D3 embryonic stem cells are positive for both Oct-3/4 and SSEA1 expression. B. Flow cytometric analysis data shows that 82.6% of mouse D3 embryonic stem cells are positive for SSEA-1 and negative for SSEA-4 a phenotype consistent with mouse embryonic stem cells. C. Flow cytometric analysis shows that mouse D3 embryonic stem cells express the pluripotent marker SOX2.

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, stem cell pluripotency is assessed by flow cytometry using the following procedure:

Fix and permeabilize cells using Fixation/Permeabilization Buffer
Stain cells with fluorochrome-conjugated antibodies or isotype controls
Wash and resuspend cells in PBS
Analyze samples by flow cytometry

View Graphic Protocol

Reagents Provided

Reagents Supplied in the Human/Mouse Embryonic Stem Cell Multi-Color Flow Cytometry Kit (Catalog # FMC001)

SOX2-PE (Clone 245610; mouse IgG_2A)
Oct-3/4-APC (Clone 240408; rat IgG_2B)
SSEA-1-PerCP (Clone MC-480; mouse IgM)
SSEA-4-CFS (Clone MC-813-70; mouse IgG₃)
Mouse IgM Isotype Control
Mouse IgG₃ Isotype Control
Rat IgG_2B Isotype Control
Mouse IgG_2A Isotype Control
Fixation/Permeabilization Buffer (with 1% formaldehyde, 8.9% saponin, and <0.05% sodium azide)
Permeabilization/Wash Buffer (with 10% saponin and 0.05% sodium azide)
Includes enough reagents to perform 25 assays

Other Supplies Required

PBS or Hanks’ Balanced Salt Solution (HBSS)

Procedure Overview

Intracellular Staining Protocol with Simultaneous Fixation/Permeabilization

Harvest cells and wash 2 times with PBS or HBSS.

Resuspend cells in Fixation/Permeabilization Buffer (approximately 5 x 10⁵ cells/0.5 mL) and transfer to 5 mL flow cytometry tubes.
Incubate cells at room temperature for 30 minutes, vortexing intermittently.

resuspend cells in buffer and transfer to flow cytometry tubes

Centrifuge samples at 300 x g for 5 minutes.
Resuspend the pellet in 100-200 μL of Permeabilization/Wash Buffer.

Add 10 µL of each fluorochrome-conjugated antibody or the corresponding isotype control antibody.
Incubate the samples at room temperature for 30-40 minutes in the dark.

add fluorchorome-conjugated antibody or isotype control antibody

Wash the cells in Permeabilization/Wash Buffer.
Resuspend the cells in 200-400 μL of PBS.

wash cells in buffer and resuspend cells in PBS

Analyze the expression of pluripotent markers simultaneously by flow cytometry.

Analyze the expression of pluripotent markers simultaneously by flow cytometry

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