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Human Pluripotent Stem Cell Functional Identification Kit

Key Product Details

Verification of Induced Pluripotent Stem Cell Pluripotency.
(5)
Analysis of Functional Tri-lineage Differentiation using Flow Cytometry.
Verification of Human Embryonic Stem Cell Pluripotency.
Verification of Human Induced Pluripotent Stem Cell Pluripotency.
Human Pluripotent Stem Cell Functional Identification Kit
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SC027B

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, the pluripotency status of human stem cells is verified using the following in vitro differentiation procedure:

  • Culture pluripotent cells of interest
  • Induce endoderm, mesoderm, and ectoderm differentiation with media supplements
  • Evaluate differentiation using germ layer markers and fluorescent ICC
 

 

Kit Components

Reagents supplied in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B):

  • Differentiation Base Media Supplement (50X)
  • Ectoderm Differentiation Supplement
  • Mesoderm Differentiation Supplement
  • Endoderm Differentiation Supplement I
  • Endoderm Differentiation Supplement II
  • Ectoderm Marker: Goat Anti-Human Otx2 Antigen Affinity-purified Polyclonal Antibody
  • Mesoderm Marker: Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal Antibody
  • Endoderm Marker: Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody
     

    Note: The quantity of each component in this kit is sufficient to make 200 mL of medium for differentiation. This is enough medium for the differentiation of one 24-well plate for each lineage.

The quantity of each component in this kit is sufficient to differentiate two 24-well plates, or an equivalent surface area, of pluripotent stem cells into hepatocyte-like cells.

 

Other Supplies Required

Reagents

  • RPMI
  • BSA, very low endotoxin
  • D-MEM/F-12 (1X)
  • GlutaMAX™ (Invitrogen or equivalent)
  • Penicillin-Streptomycin
  • Phosphate Buffered Saline (PBS)
  • Trypan Blue Solution
  • MEF Conditioned Media (Catalog # AR005 or equivalent)
  • StemXVivo Culture Matrix (100X) (Catalog # CCM013), Cultrex® PathClear® BME Reduced Growth Factor Basement Membrane Extract (Catalog # 3433-005-01), or equivalent
  • Recombinant Human FGF basic (Tissue culture grade; Catalog # 4114-TC or equivalent)
  • Accutase® (Innovative Cell Technologies or equivalent)

Materials

  • Human pluripotent stem cells
  • 24-well culture plates
  • 15 mL centrifuge tubes
  • 50 mL centrifuge tubes
  • 0.2 μm syringe filter
  • 10 mL syringe
  • Pipettes and pipette tips
  • Serological pipettes

Equipment

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

 

Procedure Overview

This protocol is designed for BG01V human embryonic stem (hES) cells and iPS2 human induced pluripotent stem (iPS) 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.

Note: The quality of the human pluripotent cells used in the differentiation is imperative. Use of suboptimal quality or very high passage pluripotent cells can result in decreased differentiation efficiency and/or increased cell death.

Undifferentiated Cell Preparation

Add 1:100 Culture Matrix or Cultrex BME in sterile PBS.

Culture Matrix or Cultrex BME in sterile PBS

Add 1.1x105 cells/cm2 in MEF Conditioned Media containing 4 ng/mL FGF Basic.

MEF Conditioned Media containing 4 ng/mL FGF Basic
 

Differentiation Procedures

 

Ectoderm
Differentiation

Mesoderm
Differentiation

Endoderm
Differentiation

Day 1 Replace media with Ectoderm Differentiation Media. Replace media with
Mesoderm Differentiation Media.
Replace media with Endoderm Differentiation Media.
Day 2 Repeat Repeat media change 12-16 hours later.
ICC detection of Brachyury (24-36 hours after initial differentiation)
16-24 hours later, Replace media with Endoderm Differentiation Media II.
Day 3 Repeat   Replace media with Endoderm Differentiation Media II.
Day 4 ICC detection of Otx2.   ICC detection of SOX17.

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Product Documents

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.