Measurement of Cytoplasmic to Nuclear Translocation

Thaddeus C. George1, Philip J. Morrissey1, Chongwei Cui2, Sukhwinder Singh2, Patricia Fitzgerald Bocarsly2

1 Amnis Corporation, Seattle, Washington, 2 University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.28
DOI:  10.1002/0471142956.cy0928s47
Online Posting Date:  January, 2009
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Abstract

A method is described for the quantitative assessment of the translocation of signaling molecules from the cytoplasm to the nucleus in cells. This method utilizes fluorochrome‐conjugated antibodies to the signaling molecule and a nuclear dye, and it is based on imagery acquired rapidly in flow with the use of a multispectral imaging cytometer. The analysis correlates the spatial distribution of the stained translocating signaling molecule with nuclear staining, and it generates a quantitative score for each cell using Pearson's correlation coefficient. Examples described in this section use reagents that detect NFκB and IRF‐7 and measure the translocation of these molecules under stimulating conditions. A protocol for combining cell surface phenotype with cytoplasm to nuclear translocation is also included. Curr. Protocol. Cytom. 47:9.28.1‐9.28.15. © 2009 by John Wiley & Sons, Inc.

Keywords: nuclear translocation; transcription factors; multispectral imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cytoplasmic to Nuclear Translocation of NFκB
  • Support Protocol 1: Image Acquisition
  • Support Protocol 2: Image Analysis
  • Alternate Protocol 1: Use of a Primary Polycolonal Anti‐NFκB Sera and A Fluorochrome‐Conjugated Secondary Reagent to Image Translocation
  • Alternate Protocol 2: Cell Surface Immunophenotyping and Assessment of IRF‐7 Translocation in Plasmacytoid Dendritic Cells
  • Support Protocol 3: PDC Enrichment
  • Support Protocol 4: Data Analysis for PDC Images
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cytoplasmic to Nuclear Translocation of NFκB

  Materials
  • THP‐1 human macrophage/monocyte line (ATCC# TIB‐202)
  • Complete RPMI 1640‐5 (see recipe)
  • Lipopolysaccharide (LPS; E. coli 0111:B4, Sigma Aldrich)
  • Wash buffer (see recipe)
  • Fixation buffer (see recipe)
  • Permeabilization buffer (see recipe)
  • Fluorochrome‐conjugated anti‐NFκB monoclonal antibody (e.g., Alexafluor488‐conjugated anti‐NFκB p65, Santa Cruz Biotechnology)
  • 1% (v/v) paraformaldehyde in phosphate‐buffered saline ( appendix 2A): 1% PFA/PBS (see recipe for fixation buffer for preparation method)
  • Nuclear dye: prodidium iodide (PI) or 7‐AAD (Sigma‐Aldrich; ImageStream channel 5, 590‐660 nm), or DRAQ5 (Biostatus Ltd.; ImageStream channel 6, 660‐730 nm)
  • 37°C, 5% CO 2 cell culture incubator
  • 15‐ml conical polypropylene centrifuge tubes (e.g., Falcon, BD)
  • Refrigerated microcentrifuge with (preferably) swinging‐bucket rotor (e.g., Beckman Coulter 22R with S241.5 rotor)
  • 1.5‐ml and 0.6‐ml siliconized snap‐cap microcentrifuge tubes (e.g., Sigma Aldrich)
  • Additional reagents and equipment for culturing (e.g., see appendix 3B) and counting ( appendix 3A) cells

Support Protocol 1: Image Acquisition

  Materials
  • Acquired images in .rif file format ( protocol 2)
  • IDEAS analytical software package (Amnis, http://www.amnis.com)

Support Protocol 2: Image Analysis

  • Polyclonal rabbit anti‐human NFκB (Santa Cruz Biotechnologies)
  • FITC‐conjugated F(ab′) 2 donkey anti‐rabbit IgG (Jackson ImmunoResearch)

Alternate Protocol 1: Use of a Primary Polycolonal Anti‐NFκB Sera and A Fluorochrome‐Conjugated Secondary Reagent to Image Translocation

  • Enriched PDC population (2–3 × 106 cells per experimental group) from human whole blood, unstimulated and stimulated with CpG‐A ( protocol 6)
  • 0.1% (v/v) bovine serum albumin/phosphate‐buffered saline ( appendix 2A; 0.1% BSA/PBS), cold
  • Pooled human sera (PHS), heat‐inactivated (30 min at 56°C)
  • PE‐conjugated anti‐BDCA‐2 (CD303) antibody (Miltenyi Biotec)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Rabbit anti‐human IRF‐7 antibody (Santa Cruz Biotechnology)
  • Goat anti‐rabbit IgG‐FITC (BioLegend, http://www.biolegend.com)
  • 5‐ml polystyrene round‐bottom tubes (BD Falcon, cat. no. 352054)

Alternate Protocol 2: Cell Surface Immunophenotyping and Assessment of IRF‐7 Translocation in Plasmacytoid Dendritic Cells

  Materials
  • Heparinized human peripheral blood
  • Ficoll‐hypaque solution (Lymphoprep; Accurate Chemical and Scientific)
  • HBSS (Hanks' buffered salt solution; appendix 2A)
  • MACS buffer (see recipe), 4°C
  • PDC isolation kit (Miltenyi Biotec) containing:
    • Non‐PDC biotin‐labeled antibody cocktail
    • Anti‐biotin magnetic microbeads
    • Separation column (LS or LD column)
  • Complete RPMI 1640‐10 (see recipe), 4°C
  • Anti‐CD123 (Biolegend) and anti‐BDCA2 (CD303; Miltenyi Biotec) antibodies
  • CpG‐A DNA oligonucleotide (TLR agonist; Coley Pharmaceutical Group)
  • Centrifuge with swinging bucket rotor
  • MACS separator magnet (Miltenyi Biotec)
  • 15‐ml conical centrifuge tubes
  • Additional reagents and equipment for counting cells ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Dai, J., Megjugorac, N.J., Amrute, S.B., and Fitzgerald‐Bocarsly, P. 2004. Regulation of IFN regulatory factor‐7 and IFN‐alpha production by enveloped virus and lipopolysaccharide in human plasmacytoid dendritic cells. J. Immunol. 173:1535‐1548.
   Deptala, A., Bedner, E., Gorczyca, W., and Darzynkiewicz, Z. 1998 Activation of nuclear factor kappa B (NF‐κB) assayed by laser scanning cytometry (LSC). Cytometry 33:376‐382.
   Fitzgerald‐Bocarsly, P., Dai, J., and Singh, S. 2008. Plasmacytoid dendritic cells and type I IFN: 50 years of convergent history. Cytokine Growth Factor Rev. 19:3‐19.
   George, T.C., Fanning, S.L., Fitzgerald‐Bocarsly, P., Medeiros, R.B., Highfill, S., Shimizu, Y., Hall, B.E., Frost, K., Basiji, D., Ortyn, W.E., Morrissey, P.J., and Lynch, D.H. 2006. Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow. J. Immunol. Methods 311:117‐129.
   Krutzik, P.O. and Nolan, G.P. 2003. Intracellular phospho‐protein staining techniques for flow cytometry: Monitoring single cell signaling events. Cytom. Part A 55:61‐70.
   Ortyn, W.E., Hall, B.F., George, T.C., Frost, K., Basiji, D.A., Perry, D.J., Zimmerman, C.A., Coder, D., and Morrissey, P.J. 2006. Sensitivity measurement and compensation in spectral imaging. Cytom. Part A 69:852‐892.
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