StemXVivo Mesoderm Kit
R&D Systems, part of Bio-Techne | Catalog # SC030B
Key Product Details
Summary for StemXVivo Mesoderm Kit
Kit Summary
For the differentiation of human pluripotent stem cells into mesoderm.
Key Benefits
- Mesoderm differentiation in 3 days
- Optimized components to reduce experimental variation
- Contains differentiation supplements and an antibody to verify mesoderm differentiation
- Involves validated and straightforward protocols
Why Differentiate Human Pluripotent Stem Cells into Mesoderm In Vitro?
Mesoderm is one of three primary germ layers formed during the process of gastrulation in the vertebrate embryo. Embryonic mesoderm gives rise to several important organs and tissues including the heart, muscle, bone, and cartilage.
Mesoderm differentiation is also an essential first step for the formation of other downstream derivative cell types, which can be used in regenerative medicine as well as toxicological and developmental studies.
The StemXVivo® Mesoderm Kit:
- Uses fully defined supplements to drive reproducible differentiation.
- Produces a healthy mesodermal cell population to increase consistency between studies and reduce unwanted experimental variability.
- Provides results in 3 days to save time and reagents.
This kit contains the following reagents to drive pluripotent stem cell differentiation into mesodermal cells and an antibody to verify differentiation status:
- Mesoderm Base Media Supplement (50x)
- Mesoderm Differentiation Supplement
- Mesoderm Marker: Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal Antibody
The quantity of each component in this kit is sufficient to make 100 mL of media for differentiation. This is enough media for the differentiation of six 60 mm plates or two 24-well plates.
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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Differentiation of Pluripotent Stem Cells into Mesoderm. Human embryonic stem cells were differentiated into mesoderm using the media supplements included in this kit. To evaluate lineage commitment, the cells were stained with the Goat Anti-Human Brachyury Antibody included. The cells were stained using the NorthernLights 557-conjugated Donkey anti-Goat IgG Secondary Antibody (Catalog # NL001; red), and the nuclei were counterstained with DAPI (blue). |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, human pluripotent stem cells are differentiated into mesoderm cells using the following mesoderm differentiation procedure:
- Plate cells on coated plates
- Replace MEF Conditioned Media with Differentiation Media
- Evaluate differentiation status using the included Brachyury antibody
- Cells are ready for downstream applications on Day 3
Reagents Provided
Reagents supplied in the StemXVivo® Mesoderm Kit (Catalog # SC030B).
- Mesoderm Base Media Supplement (50X)
- Mesoderm Differentiation Supplement
- Mesoderm Marker: Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal Antibody
Other Supplies Required
Reagents
- RPMI Medium 1640
- BSA, very low endotoxin
- DMEM/F-12
- GlutaMAX™ (Invitrogen), or equivalent
- Penicillin-Streptomycin (optional)
- Phosphate-Buffered Saline (PBS)
- Cultrex® Basement Membrane Extract Reduced Growth Factor, PathClear®(Catalog # 3433-005-01)
- Recombinant Human FGF basic (Tissue culture grade; Catalog # 4114-TC)
- MEF Conditioned Media (Catalog #AR005)
- Trypan Blue solution
- Accutase® (Innovative Cell Technologies), or equivalent
- 95% Ethanol
- 4% Paraformaldehyde in PBS
- 1% BSA
- 0.3% Triton™ X-100, 1% BSA, 10% normal donkey serum in PBS
- Mounting medium (Catalog # CTS011)
- Secondary developing reagents (Catalog # NL001, NL002, or NL003)
- Sterile, deionized water
Materials
- Human pluripotent stem cells
- 24-well culture plates
- 12 mm cover slips
- 15 mL centrifuge tubes
- 50 mL centrifuge tubes
- 0.2 μm syringe filter
- 0.2 μm, 500 mL filter units
- 10 mL syringes
- Serological pipettes
- Pipettes and pipette tips
- Glass slides
- Fine pointed curved forceps
Equipment
- 37 °C and 5% CO2 incubator
- 37 °C water bath
- Centrifuge
- Hemocytometer
- Inverted microscope
- Fluorescence microscope
Precaution: The acute and chronic effects of overexposure to reagents of this kit are unknown. Safe laboratory procedures should be followed and protective clothing should be worn when handling kit reagents.
Some components of this kit contain sodium azide, which may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.
Procedure Overview
This protocol is designed for BG01V human embryonic stem cells grown in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog #AR005). If using different cell lines or growth media, this protocol may need to be optimized.
Coat wells with Cultrex® Basement Membrane Extract.
Incubate at room temperature for 1-2 hours.
Plate human pluripotent stem cells onto the coated plates at 1.1 x 105 cells/cm2 in MEF Media containing FGF basic.
Culture the cells overnight at 37 °C and 5% CO2. The next day each well should be approximately 50% confluent.
Day 1 of Differentiation
Remove the MEF Conditioned Media with Mesoderm Differentiation Media.
Incubate at 37 °C and 5% CO2 for 12-16 hours.
Day 2 of Differentiation
Repeat media change.
After 12-20 hours, the cells are ready for further differentiation to downstream cell types or analysis by immunocytochemistry and/or flow cytometry.
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