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StemXVivo Endoderm Kit

For endoderm differentiation of human pluripotent stem cells

Key Product Details

Definitive Endoderm Differentiation of BG01V Human Embryonic Stem Cells.
(5)
The Differentiation of BG01V Human Embryonic Stem Cells into Definitive Endoderm Confirmed by Cell Markers (SOX17+/CXCR4+/FOX2A+/SOX7low). 
Differentiation of Human Amniotic Fluid Stem Cells into Definitive Endoderm.
Expression of SOX17 and HNF-3 beta in Human Amniotic Fluid Stem Cells Differentiated into Definitive Endoderm.
StemXVivo Endoderm Kit
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SC019B

Assay Procedure

Refer to the product datasheet for complete product details.

 

Briefly, human pluripotent stem cells are differentiated into endoderm cells using the following endoderm differentiation procedure:

  • Plate cells on coated plates
  • Replace MEF Conditioned Media with Differentiation Media
  • Evaluate differentiation status using the included SOX17 antibody
  • Cells are ready for downstream applications on Day 4
 

Reagents Provided

Reagents supplied in the StemXVivo® Endoderm Kit (Catalog # SC019B):

  • Endoderm Base Media Supplement (50x)
  • Recombinant Human Activin A
  • Recombinant Human FGF Basic
  • Recombinant Human Wnt-3a
  • Endoderm Marker: Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody

Other Supplies Required

Reagents

  • Human pluripotent stem cells
  • RPMI Medium 1640
  • DMEM/F-12
  • GlutaMAX (Invitrogen), or equivalent
  • Penicillin-Streptomycin
  • Phosphate-Buffered Saline (PBS)
  • Cultrex® Basement Membrane Extract Reduced Growth Factor, PathClear®(Catalog # 3433-005-01) or StemXVivo® Culture Matrix (Catalog # CCM013)
  • MEF Conditioned Media (Catalog # AR005)
  • Trypan Blue Stain
  • Accutase® (Innovative Cell Technologies), or equivalent
  • BSA, very low endotoxin
  • Sterile, deionized water
 

Materials

  • 60 mm tissue culture dishes
  • 24-well culture plates
  • 15 mL centrifuge tubes
  • 50 mL centrifuge tubes
  • 0.2 mm syringe filter
  • 0.2 mm, 500 mL filter units
  • 10 mL syringes
  • Serological pipettes
  • Pipettes and pipette tips
 

Equipment

  • 37 °C and 5% CO2 incubator
  • 37 °C water bath
  • Centrifuge
  • Hemocytometer
  • Inverted microscope
 

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 (Catalog # 3432-005-01) or StemXVivo® Culture Matrix (Catalog # CCM013).

 

Incubate at room temperature for 1-2 hours.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 1

Plate BG01V human embryonic stem cells onto the coated plates at 3.3 x 103 cells/cm2 in MEF Media containing FGF basic.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 2

Culture the cells overnight at 37 °C and 5% CO2. The next day each well should contain a tightly packed monolayer of cells

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 3

Day 1 of Differentiation

 

Remove the MEF Conditioned Media from the wells 12-24 hours after initial plating.

Add fresh MEF Conditioned Media containing FGF basic.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 4

Incubate at 37 °C and 5% CO2 for a minimum of 2-4 hours prior to adding Differentiation Media I.

Remove the MEF Conditioned Media from each well.

Wash each well once with once with 1X PBS.

Add Differentiation Media I to each well and incubate overnight at 37 °C and 5% CO2.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 5

Day 2 and 3 of Differentiation

 

Approximately 12-16 hours after adding Differentiation Media I, replace the media with Differentiation Media II.

Incubate Continue to replace Differentiation Media II every 8-12 hours.

Day 4 of Differentiation

 

On Day 4, the cells are ready for further differentiation to downstream cell types or analysis by immunocytochemistry and/or flow cytometry.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 5

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Product Documents

Certificate of Analysis

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