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StemXVivo Cardiomyocyte Differentiation Kit

Bio-Techne includes R&D Systems | Catalog # SC032B

This item is in process of being discontinued.

Key Product Details

iPSC-Derived Cardiomyocyte Contractions Visualized Using the Calcium Indicator, Fluo-4. 
(6)
Differentiation of Pluripotent Stem Cells into Cardiomyocytes. 
Improved Efficiency of iPSC Differentiation Across Pluripotent Stem Cell Lines. 
Endothelin-1 Induces Expression of Atrial Natriuretic Peptide in Cardiomyoctyes Derived from iPSCs Using the New Kit.
Kit-Derived Cardiomyocytes Express Atrial and Ventricular Markers. 
Kit-Derived Cardiomyocytes Express Stage-specific Markers During Differentiation.

Assay Procedure

Refer to the product datasheet for complete product details.

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

  • Plate cells on coated plates
  • Replace MEF Conditioned Media with Cardiomyocyte Differentiation Base Media I containing RGF BME
  • Replace the media with Day 1 Differentiation Media
  • Replace the media with Day 5 Differentiation Media
  • Replace the media with Cardiomyocyte Differentiation Base Media II
  • Evaluate differentiation status using the included Troponin T antibody
  • Cells are ready for downstream applications
 

 

Reagents Provided

Reagents supplied in the StemXVivo® Cardiomyocyte Differentiation Kit (Catalog # SC032).

  • Stem Cell Qualified RGF BME, Pathclear®
  • Cardiomyocyte Differentiation Base Media Supplement I
  • Cardiomyocyte Differentiation Base Media Supplement II
  • Cardiomyocyte Differentiation Cocktail I
  • Cardiomyocyte Differentiation Cocktail IIA
  • Cardiomyocyte Differentiation Cocktail IIB
  • Cardiomyocyte Differentiation Cocktail III
  • Anti-Human Cardiac Troponin T Antibody

 

Other Supplies Required

Reagents

  • RPMI 1640
  • BSA, very low endotoxin
  • D-MEM/F-12 (1X)
  • GlutaMAX (Invitrogen, Catalog # 35050-079 or equivalent)
  • Penicillin-Streptomycin (optional)
  • Phosphate Buffered Saline (PBS)
  • 95% Ethanol
  • 4% Paraformaldehyde
  • 1% BSA in PBS
  • 0.3% Triton X-100, 1% BSA, 10% normal donkey serum in PBS
  • Mounting medium (R&D Systems, Catalog # CTS011)
  • Secondary developing reagent (R&D Systems, Catalog # NL001)
  • Deionized or distilled water

Materials

  • Human pluripotent stem cells
  • 24-well culture plates (or other, as needed)
  • 60 mm culture plates
  • 12 mm coverslips (optional)
  • 15 mL and 50 mL centrifuge tubes
  • 0.2 μm syringe filter
  • 10 mL syringe
  • Pipettes and pipette tips
  • Serological pipettes
  • Glass slides
  • Fine pointed curved forceps
  • FACS tubes
  • Flow Cytometry Fixation/Permeabilization Buffer I (R&D Systems, Catalog # FC007) supplemented with 0.1% Triton X-100
  • Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems, Catalog # FC005)

Equipment

  • 37 °C and 5% CO2 incubator
  • 37 °C water bath
  • Centrifuge
  • 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.

 

Procedure Overview

This protocol has been tested on human pluripotent stem cells cultured in either MEF Conditioned Media (R&D Systems, Catalog # AR005) or equivalent. The quality and differentiation potential of human pluripotent stem cells at the onset of the differentiation protocol are of paramount importance to the efficiency of differentiation. Human pluripotent stem cells must be > 95% positive for OCT-3/4.

Coat wells with Stem Cell Qualified PathClear® RGF BME (RGF BME).

Incubate at room temperature for 1-2 hours.

SC032B Step 1

Plate human pluripotent stem cells onto the coated plates at 3-4 x 104 cells/cm2 in Pluripotent Stem Cell Maintenance Media.

Culture cells to 80-90% confluency.

SC032B Step 2

Day (-1) of Differentiation

Replace the stem cell culture media with ice cold Pluripotent Stem Cell Maintenance Media containing RGF BME diluted 1:60.

Incubate at 37 °C and 5% CO2 for 18-24 hours.

SC032B Step 3

Day 0 of Differentiation

Replace the media with ice cold Day 0 Cardiomyocyte Differentiation Media containing RGF BME diluted 1:60.

Incubate at 37 °C and 5% CO2 for 24 hours.

SC032B Step 4

Day 1 of Differentiation

Replace the media with Day 1 Cardiomyocyte Differentiation Media

Incubate at 37 °C and 5% CO2 for 4 DAYS without media exchange.

SC032B Step 5

Day 5 of Differentiation

Replace the media with Day 5 Cardiomyocyte Differentiation Media.

Incubate at 37 °C and 5% CO2 for 2 DAYS without media exchange.

SC032B Step 6

Day 7 of Differentiation and Beyond

Replace the media with Cardiomyocyte Differentiation Base Media I.

Incubate at 37 °C and 5% CO2. Replace media every 1-2 days as needed.

SC032B Step 7

Day 12 of Differentiation and Beyond

Replace the media with Cardiomyocyte Differentiation Base Media II.

Incubate at 37 °C and 5% CO2. Replace media every 1-2 days as needed.

SC032B Step 8

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FAQs for StemXVivo Cardiomyocyte Differentiation Kit

  • Can the StemXVivo Cardiomyocyte Differentiation Kit, Catalog # SC032B, use both embryonic stem cells (ESCs) and iPSCs as the starting cell population?

    Yes, SC032B can be used with both ESCs and iPSCs. 

Product Documents for StemXVivo Cardiomyocyte Differentiation Kit

Certificate of Analysis

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