H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit
R&D Systems, part of Bio-Techne | Catalog # FMC001
Key Product Details
Summary for H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit
Kit Summary
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.
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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. |
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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. |
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
Reagents Provided
Reagents Supplied in the Human/Mouse Embryonic Stem Cell Multi-Color Flow Cytometry Kit (Catalog # FMC001)
- SOX2-PE (Clone 245610; mouse IgG2A)
- Oct-3/4-APC (Clone 240408; rat IgG2B)
- 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 IgG2B Isotype Control
- Mouse IgG2A 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.
- 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.
- Wash the cells in Permeabilization/Wash Buffer.
- Resuspend the cells in 200-400 μL of PBS.
- Analyze the expression of pluripotent markers simultaneously by flow cytometry.
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