Quantification of Pluripotency Transcription Factor Levels in Embryonic Stem Cells by Flow Cytometry

Nicola Festuccia1, Ian Chambers1

1 Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, United Kingdom
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.9
DOI:  10.1002/9780470151808.sc01b09s19
Online Posting Date:  December, 2011
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Abstract

Embryonic stem (ES) cell lines are derived from the inner cell mass of the pre‐implantation blastocyst and are characterized by the ability to undergo indefinite self‐renewal while retaining the potential to differentiate into each of the three primary germ layers. The ability of individual ES cells to self‐renew or appropriately respond to differentiation signals is influenced by the intracellular level of a number of crucial transcription factors. It is therefore important to be able to reliably quantify the levels of these proteins in single cells. Here we present an intracellular staining technique for flow cytometry suitable for monitoring transcription factor expression in ES cells. We illustrate the application of this technique to the detection of Oct4 and Nanog proteins and the coupling of this approach with fluorescent reporters of gene activity. Curr. Protoc. Stem Cell Biol. 19:1B.9.1‐1B.9.13. © 2011 by John Wiley & Sons, Inc.

Keywords: ES cells; FACS; quantitation; intracellular staining; Nanog Oct4

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • ES cells grown in 25‐cm2, 75‐cm2, or 125‐cm2 gelatinized flasks (as described in Smith, )
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • 0.025% (v/v) Trypsin (see recipe)
  • Complete ES cell medium (see recipe)
  • Paraformaldehyde (PFA; see recipe)
  • 70% (v/v) methanol (see recipe)
  • PBS containing 5% (v/v) serum from the same species as the secondary antibody is raised in (e.g., goat serum)
  • Staining buffer (see recipe)
  • Appropriate antibodies (monoclonal anti‐Oct4 and polyclonal anti‐Nanog antibodies at a final concentration of 2.5 µg/ml and 10 µg/ml, respectively, are used in this protocol)
  • Appropriate secondary antibodies (anti‐mouse Alexa fluor‐647 antibody at a final concentration of 4 µg/ml and anti‐rabbit Alexa fluor‐405 antibody at a final concentration of 10 µg/ml are used in this protocol)
  • Universal 30‐ml tubes (Sterilin, cat. no. 128A)
  • Centrifuge
  • 5‐ml polystyrene round‐bottom tubes (BD Falcon, cat. no. 352054)
  • Denley Spiramix 5 tube roller (Thermo, cat. no. DS507) or SB2 rotator (Stuart, cat. no. SB2)
  • Microtiter plates 96‐well/V‐bottom (Sterilin, cat. no. 612V96)
NOTE: Antibodies used to generate data shown in this paper are listed in Table 1.9.1. Additional antibodies useful for monitoring pluripotency transcription factor expression are listed in Table 1.9.2.
Table 1.0.1   MaterialsSources and Suggested Concentrations of Antibodies Used in this StudyAdditional Antibodies That Have Been Tested for Use in the Described Protocol

Antibody Supplier Catalog number Suggested concentration
Mouse monoclonal anti‐Oct3/4 (C‐10) Santa Cruz Biotechnology SC‐5279 2.5 µg/ml
Rabbit polyclonal anti‐Nanog Abcam Ab14959 10 µg/ml
Goat anti‐mouse IgG (H+L) Alexa fluor‐647 Invitrogen, Molecular Probes A21235 4 µg/ml
Goat anti‐rabbit IgG (H+L) Alexa fluor‐405 Invitrogen, Molecular Probes A31556 10 µg/ml
Antibody Supplier Catalog number Suggested concentration
Mouse monoclonal anti‐Klf4 Abcam Ab75486 10 µg/ml
Mouse monoclonal anti‐human Esrrb (cross‐reacts with mouse) Perseus Proteomics PP‐H6707H 10 µg/ml
Goat polyclonal anti‐Oct3/4 (N‐19) Santa Cruz Biotechnology SC‐8628 1.25 µg/ml
Donkey anti‐mouse IgG (H+L) Alexa fluor‐647 Invitrogen, Molecular Probes A21235 4 µg/ml
Donkey anti‐goat IgG (H+L) Alexa fluor‐568 Invitrogen, Molecular Probes A21235 4 µg/ml
Donkey anti‐rabbit IgG biotin Millipore AP182B 10 µg/ml
Streptavidin Alexa fluor‐405 Invitrogen, Molecular Probes S32351 4 µg/ml

Table 1.0.2   MaterialsSources and Suggested Concentrations of Antibodies Used in this StudyAdditional Antibodies That Have Been Tested for Use in the Described Protocol

Antibody Supplier Catalog number Suggested concentration
Mouse monoclonal anti‐Oct3/4 (C‐10) Santa Cruz Biotechnology SC‐5279 2.5 µg/ml
Rabbit polyclonal anti‐Nanog Abcam Ab14959 10 µg/ml
Goat anti‐mouse IgG (H+L) Alexa fluor‐647 Invitrogen, Molecular Probes A21235 4 µg/ml
Goat anti‐rabbit IgG (H+L) Alexa fluor‐405 Invitrogen, Molecular Probes A31556 10 µg/ml
Antibody Supplier Catalog number Suggested concentration
Mouse monoclonal anti‐Klf4 Abcam Ab75486 10 µg/ml
Mouse monoclonal anti‐human Esrrb (cross‐reacts with mouse) Perseus Proteomics PP‐H6707H 10 µg/ml
Goat polyclonal anti‐Oct3/4 (N‐19) Santa Cruz Biotechnology SC‐8628 1.25 µg/ml
Donkey anti‐mouse IgG (H+L) Alexa fluor‐647 Invitrogen, Molecular Probes A21235 4 µg/ml
Donkey anti‐goat IgG (H+L) Alexa fluor‐568 Invitrogen, Molecular Probes A21235 4 µg/ml
Donkey anti‐rabbit IgG biotin Millipore AP182B 10 µg/ml
Streptavidin Alexa fluor‐405 Invitrogen, Molecular Probes S32351 4 µg/ml

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Figures

Videos

Literature Cited

Literature Cited
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