StemXVivo Mesoderm Kit
R&D Systems, part of Bio-Techne | Catalog # SC030B
Key Product Details
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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 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
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.
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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