Whole Blood Processing for Measurement of Signaling Proteins by Flow Cytometry

Sue Chow1, David Hedley1, T. Vincent Shankey2

1 Division of Applied Molecular Oncology, Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Ontario, Canada, 2 Advanced Technology Center, Beckman Coulter, Inc., Miami, Florida
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.27
DOI:  10.1002/0471142956.cy0927s46
Online Posting Date:  October, 2008
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Abstract

Signal transduction pathways link external stimuli with cellular responses, which normally regulate cell proliferation, death, and differentiation. The study of signal transduction was revolutionized through the development of phospho‐specific antibodies that recognize proteins only when they are phosphorylated at specific sites. As discussed by Nolan and co‐workers (UNIT), one of the unique features of flow cytometry is its ability to perform correlated measurements of multiple phosphorylation states at the single cell level. This provides insight into the complexity of signaling networks that is not obtained by standard biochemical techniques. Furthermore, in combination with other phenotypic markers, flow cytometry can measure alterations in signaling pathways in subpopulations of cells. This clearly has wide potential for studying disorders of the hematopoietic and immune systems. Curr. Protocol. Cytom. 46:9.27.1‐9.27.19. © 2008 by John Wiley & Sons, Inc.

Keywords: whole blood fixation/permeabilization; signal transduction; phospho‐specific antibody; pharmacodynamics; human leukemias

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Whole Blood Processing for Measurement of Signaling Proteins by Flow Cytometry
  • Alternate Protocol 1: Measurement of Signaling Proteins by Flow Cytometry using Frozen, Fixed Cells
  • Alternate Protocol 2: Alcohol Unmasking of Phospho‐Epitopes
  • Basic Protocol 2: Staining Cell Surface Markers (CD) Before Fixation and Permeabilization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Whole Blood Processing for Measurement of Signaling Proteins by Flow Cytometry

  Materials
  • Anticoagulated blood or bone marrow in appropriate tubes: e.g., 6‐ml lithium heparin vacutainer tubes (BD Biosciences, cat. no. 367886) or 3‐ml K 2EDTA vacutainer tubes (BD Biosciences, cat. no. 367856)
  • Inhibitor working solutions, for example:
    • 10 mM U0126 (MEK 1/2 inhibitor; Cell Signaling Technology) in 100% absolute methanol; store up to 6 months at −20°C
    • 50 mM Ly 294002 (Calbiochem) in 100% anhydrous ethanol: store up to 6 months at −20°C
    • 0.1 mg/ml rapamycin solution (see recipe)
  • Agonist working solutions, for example
    • 40 µM phorbol myristate acetate (PMA; Sigma) in 100% anhydrous ethanol (store up to 1 year at −20°C)
    • Stem cell factor (SCF) working solution (see recipe)
  • 10% (v/v) formaldehyde, methanol free (Polysciences): store up to 6 months at room temperature in the dark
  • Lysis/permeabilization buffer (see recipe), 37°C
  • Wash buffer: 4% (v/v) fetal bovine serum (FBS) in Dulbecco's phosphate‐buffered saline, calcium and magnesium free (CMF‐DPBS): sterilize by passing through a 0.22‐µm filter and store at 4°C
  • Freezing medium (see recipe), optional
  • Antibodies (conjugated to fluorescent labels) to CD (cell surface) markers (see ), for example:
    • CD45‐PE Cy7 (clone J.33; Beckman Coulter)
    • CD3‐PE (clone UCHT1, Beckman Coulter)
    • CD34‐PE (clone 581; Beckman Coulter)
    • CD45‐PE (clone J.33; Beckman Coulter)
    • CD117‐PE (clone 104D2D1; Beckman Coulter)
  • Antibodies (conjugated with fluorescent labels) to intracellular phospho‐epitopes (see ), for example:
    • p‐ERK (T202/Y204) clone E10‐Alexa 488 or ‐Alexa 647 (Beckman Coulter)
    • p‐Akt (S473) clone 193H12‐Alexa 488 or ‐Alexa 647 (Cell Signaling Technology)
    • p‐S6 (S235/S236; Cell Signaling Technology)
  • Diluent buffer: 4% (v/v) FBS/0.1% (w/v) NaN 3 /CMF‐DPBS
  • Analysis buffer: 0.1% (w/v) paraformaldehyde/CMF‐DPBS
  • 12 × 75–mm polypropylene tubes and racks
  • 6‐in. cotton‐tip swabs
  • Repeater pipettor, recommended
  • Flow cytometer

Alternate Protocol 1: Measurement of Signaling Proteins by Flow Cytometry using Frozen, Fixed Cells

  • Frozen, fixed cells ( protocol 1, step 15a)

Alternate Protocol 2: Alcohol Unmasking of Phospho‐Epitopes

  • 50% methanol/0.85% NaCl: store at −20°C

Basic Protocol 2: Staining Cell Surface Markers (CD) Before Fixation and Permeabilization

  Materials
  • Anticoagulated blood or bone marrow in appropriate tubes: e.g., 6‐ml lithium heparin vacutainer tubes (BD Biosciences) and 3‐ml K 2EDTA vacutainer tubes (BD Biosciences)
  • Antibodies (conjugated to fluorescent labels) to CD (cell surface) markers (see ), for example:
    • CD3‐PE (clone UCHT1; Beckman Coulter)
    • CD34‐PE (clone 581; Beckman Coulter)
    • CD45‐PE (clone J.33; Beckman Coulter)
    • CD117‐PE (clone 104D2D1; (Beckman Coulter)
  • Inhibitor working solutions, for example:
    • 10 mM U0126 (MEK 1/2 inhibitor; Cell Signaling Technologies) in 100% absolute methanol; store up to 6 months at −20°C
    • 50 mM Ly 294002 (Calbiochem) in 100% anhydrous ethanol; store up to 6 months at −20°C
    • 1 mg/ml rapamycin solution (see recipe)
  • Agonist working solutions, for example:
    • 40 µM phorbol myristate acetate (PMA; Sigma) in 100% anhydrous ethanol; store up to 1 year at −20°C
    • Stem cell factor (SCF) working solution (see recipe)
  • 10% (v/v) formaldehyde, methanol free (Polysciences): store up to 6 months at room temperature in the dark
  • Lysis/permeabilization buffer (see recipe), 37°C
  • Wash buffer: 4% (v/v) fetal bovine serum (FBS) in Dulbecco's phosphate‐buffered saline, calcium and magnesium free (CMF‐DPBS): sterilize by passing through a 0.22‐µm filter and store at 4°C
  • Freezing medium (see recipe), optional
  • Antibodies (conjugated to fluorescent labels) to intracellular phospho‐epitopes (see ), for example:
    • p‐ERK (T202/Y204) clone E10‐Alexa 488 or ‐Alexa 647 (Beckman Coulter)
    • p‐Akt (S473) clone 193H12‐Alexa 488 or ‐Alexa 647 (Cell Signaling Technology)
    • p‐S6 (S235/S236; Cell Signaling Technology)
  • Diluent buffer: 4% (v/v) FBS/0.1% (w/v) NaN 3 /CMF‐DPBS
  • Analysis buffer: 0.1% (w/v) paraformaldehyde/CMF‐DPBS
  • 12 × 75–mm polypropylene tubes and racks
  • 6‐in. cotton‐tip swabs
  • Repeater pipettor, recommended
  • Flow cytometer
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Figures

Videos

Literature Cited

Literature Cited
   Chow, S., Patel, H., and Hedley, D.W. 2001. Measurement of MAP kinase activation by flow cytometry using phospho‐specific antibodies to MEK and ERK: Potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry 46A:72‐78.
   Chow, S., Hedley, D.W., Grom, R., Magari, R., Jacobberger, J., and Shankey, T.V. 2005. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry 67A:4‐17.
   Chow, S., Minden, M.D., and Hedley, D.W. 2006. Constitutive phosphorylation of the S6 ribosomal protein via mTOR and ERK signaling in the peripheral blasts of acute leukemia patients. Exp. Hematol. 34:1182‐1190.
   Jacobberger, J.W. 2000. Flow cytometric analysis of intracellular protein epitopes. In Immunophenotyping (C. Stewart and J. Nickolson, eds.) pp. 361‐406. Wiley‐Liss, New York.
   Jacobberger, J.W., Sramkoski, R.M., Frisa, P.S., Peng Ye, P., Gottlieb, M.A., Hedley, D.W., Shankey, T.V., Smith, B.L., Paniagua, M., and Goolsby, C.L. 2003. Immunoreactivity of STAT5 phosphorylated on tyrosine 694 as a cell‐based measure of Bcr/Abl kinase activity. Cytometry 54A:75‐88.
   Krutzik, P.O. and Nolan, G.P. 2003. Intracellular phosphoprotein staining techniques for flow cytometry: Monitoring single cell signaling events. Cytometry 55A:61‐70.
   Shankey, T.V., Forman, M., Scibelli, P., Cobb, J., Smith, C.M., Mills, R., Bernal‐Hoyos, E., Van Der Heiden, M., Popma, J., and Keeney, M. 2006. An optimized whole blood method for flow cytometric measurement of ZAP‐70 protein expression in chronic lymphocytic leukemia. Cytometry 70B:259‐269.
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