Early Events in B Lymphocyte Activation

Subbarao Bondada1, Amy Troyer1, Ralph L. Chelvarajan1

1 University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.9
DOI:  10.1002/0471142735.im0309s57
Online Posting Date:  November, 2003
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B cell activation can be quantitated indirectly by assaying antibody production or directly by measuring cellular changes that occur immediately after exposure to an activation signal. This unit provides methods for the latter (direct) approach‐‐namely, methods for quantifying early parameters of B cell activation such as increases in intracellular ionized calcium concentration [Ca2+]I, cell size, and MHC class II‐antigen expression. These assays are not so much B cell specific as activation specific; as such they are a useful means of providing information about cellular events that are independent of antibody secretion. While the protocols included in this unit have been specifically designed for use with murine B cells, they can also be used with B cells obtained from other species. In addition, the method described here also applies to T cells.

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Table of Contents

  • Basic Protocol 1: Bcr‐Induced Changes in Intracellular Calcium in B Cells
  • Basic Protocol 2: Analysis of Protein Tyrosine Phosphorylation
  • Basic Protocol 3: Analysis of Cell Size and Expression of Molecules Indicative of B Cell Activation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Bcr‐Induced Changes in Intracellular Calcium in B Cells

  • Gey's solution (see recipe), 4°C
  • Hanks balanced salt solution (HBSS; appendix 2A)/5% FBS
  • IF12 or RPMI medium (unit 3.8) containing 10% FBS
  • DMSO, anhydrous
  • INDO‐1 AM (Molecular Probes)
  • FITC‐anti‐B220 (Sigma or BD PharMingen)
  • HBSS ( appendix 2A), 4°C
  • 1.0 mM ionomycin (Calbiochem)
  • F(ab′) 2 anti‐IgM (µ chain specific; ICN Biomedicals) or anti‐IgD (BD PharminGen or Southern Biotechnology)
  • 12 × 75–mm tubes with caps suitable for use on the given flow cytometer
  • Flow cytometer (i.e., BD PharMingen FACS Vantage, Beckman‐Coulter Cytomics FC‐500 or EPICS, Cytomation Mo‐Flo, or any instrument that has a laser to excite Indo‐1 in the UV range, and FITC and phycoerythrin in the visible range)
  • Additional reagents and equipment for preparation of murine spleen cells (unit 3.4) and calculating calcium concentration (unit 5.5)

Basic Protocol 2: Analysis of Protein Tyrosine Phosphorylation

  • Serum‐free IF12 or RPMI medium (unit 3.8)
  • 25 µg/ml F(ab′) 2 anti‐IgM (µ‐chain specific; ICN Biomedicals)
  • 1× PBS, pH 7.2 ( appendix 2A), with and without phosphatase inhibitors (i.e., 10 mM NaF and 1 mM Na 3VO 4), 4°C
  • Lysis buffer (see recipe)
  • BCA Protein Assay reagent (Pierce)
  • 1.5% BSA in 1× TBST (see recipe)
  • RC20:HRPO (BD PharMingen, Transduction Laboratories) or HRP‐conjugated 4G10 (Upstate) anti‐phosphotyrosine antibody in 1.5% BSA/1× TBST
  • 1× TBST (see recipe)
  • Enhanced chemiluminescence kit (Perkin‐Elmer Life Sciences or Pierce)
  • Immobilon‐P membrane (Millipore)
  • Additional reagents and equipment for purifying B lymphocytes depleted of red blood cells (see protocol 1 and unit 3.8), SDS‐PAGE (unit 8.4), protein blotting (unit 8.10), autoradiography ( appendix 3J), and immunoprecipitation (unit 8.3)

Basic Protocol 3: Analysis of Cell Size and Expression of Molecules Indicative of B Cell Activation

  • Purified resting B cells (unit 3.8)
  • IF12 or RPMI (unit 3.8) with 5% FBS
  • 25 µg/ml F(ab′) 2 anti‐µ (ICN Biomedicals), 1.0 µg/ml anti‐CD40 (clone 1C1D; Southern Biotechnology or eBioscience), or 1.0 µg/ml LPS (Sigma)
  • FACS buffer (see recipe)
  • 10 mg/ml 2.4G2 anti‐Fc receptor antibody (BD PharMingen)
  • FITC‐anti‐mouse‐MHC class II (clones MKD6, 14.4.4 and M5/114.5.2), ‐CD80, ‐CD86, or ‐CD138 (BD PharMingen, Sigma, or equivalent)
  • 1× PBS ( appendix 2A)
  • Fixing solution (see recipe; optional)
  • 96‐, 48‐, or 24‐well plates
  • Additional reagents and materials for FACS analysis (Chapter 5)
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Literature Cited

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