Analysis of Immunotoxicity by Enumeration of Antibody‐Producing B Cells

Stacey E. Anderson1, Albert E. Munson1, Barbara J. Meade1

1 National Institute for Occupational Safety and Health, Morgantown, West Virginia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.11
DOI:  10.1002/0471140856.tx1811s29
Online Posting Date:  September, 2006
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Abstract

The plaque‐forming cell (PFC) assay measures the humoral immune response mediated by the concerted actions of antigen‐presenting cells, T lymphocytes, and B lymphocytes. The most common form of the plaque method is used for the detection of murine primary IgM antibodies directed against the T cell–dependent sheep red blood cell (sRBC) surface antigens. Research has shown that the PFC response to sRBC is not only an excellent monitor of the primary effector function of the B cell, but that it may be the most sensitive immune parameter currently available to identify chemical perturbation. Several modifications and variations of the PFC assay are described in detail in this unit.

Keywords: Immunotoxicity; humoral immunity; antibody‐forming cells

     
 
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Table of Contents

  • Basic Protocol 1: The Plaque‐Forming Cell (PFC) Response to Sheep Red Blood Cells In Vivo
  • Alternate Protocol 1: Mishell‐Dutton Assay: In Vitro Plaque Forming Cell Response to Sheep Red Blood Cells
  • Alternate Protocol 2: In Vivo Chemical Exposure/In Vitro PFC Response to sRBC
  • Alternate Protocol 3: In Vitro Plaque‐Forming Cell Response TO sRBC with a Metabolic Activation System
  • Alternate Protocol 4: Measuring Plaque‐Forming Cell Response to T‐Independent Antigens
  • Support Protocol 1: Preparation of sRBC for Immunization
  • Support Protocol 2: Preparation of Haptenated sRBC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Plaque‐Forming Cell (PFC) Response to Sheep Red Blood Cells In Vivo

  Materials
  • Male and/or female mice: typically B6C3F1 female mice (Taconic Farms) are used for this assay (Koller and Exon, )
  • Potentially immunotoxic chemical for testing along with vehicle control and positive control (typically, cyclophosphamide)
  • HBSS/HEPES (see recipe)
  • 3.75 × 108 cell/ml suspension of sheep red blood cells (sRBC) in HBSS, pH 7.0 ( protocol 6)
  • Bacto Agar (BD Biosciences)
  • 30 mg/ml DEAE‐dextran (prepared from dextran of average mol. wt. of 500,000; Sigma) in 0.9% (w/v) NaCl (adjust pH to 6.9 when dissolved; store up to 4 months at 4°C)
  • 70% ethanol
  • Guinea pig complement (GPC), lyophilized (Cedarlane Laboratories; http://www.cedarlanelabs.com)
  • 50% (v/v) suspension of sRBC (unprocessed, from same sheep donor/blood stock as sRBC used for immunization) in Alsever's solution (see recipe for Alsever's solution)
  • Hanks' balanced salt solution (HBSS), pH 7.0 (see recipe)
  • Isoton (Beckman Counter)
  • Zap‐O‐Globin II (Beckman Coulter)
  • 12 × 75–mm disposable borosilicate snap‐cap glass tubes and corresponding racks
  • 1‐ml syringes with 25‐G 5/8‐in. needles
  • 100 × 15–mm polystyrene petri dishes
  • 250‐ml Pyrex Erlenmeyer flask
  • 43° to 45°C water bath
  • Surgical instruments: scissors and forceps
  • 5‐ml plastic capped tubes
  • Balance (accurate to ± 0.5 mg)
  • 60 × 15–mm petri dishes
  • Frosted microscope slides
  • Refrigerated centrifuge
  • 15‐ml conical centrifuge tubes
  • 45 × 50–mm cover slips (each weighing ∼750 mg; Fisher)
  • 36° to 38°C incubator with thermometer
  • Plaque viewer (Bellco Biotechnology) or inverted microscope
  • Accuvette II vials (Beckman Coulter)
  • Coulter Counter (Beckman Coulter)
  • Additional reagents and equipment for intravenous (i.v.) injection of the mouse (Donovan and Brown, ), euthanasia of the mouse by CO 2 asphyxiation (Donovan and Brown, ), and counting cells ( appendix 3B)

Alternate Protocol 1: Mishell‐Dutton Assay: In Vitro Plaque Forming Cell Response to Sheep Red Blood Cells

  • Complete RPMI medium (see recipe)
  • 50% (v/v) suspension of sRBC (unprocessed, from original sheep donor) in Alsever's solution (see recipe for Alsever's solution)
  • Blood gas mixture: 7% CO 2/10% O 2/83% N 2
  • DEAE‐dextran (powder)
  • 48‐well tissue culture plates, sterile
  • Mishell‐Dutton pressure box (CBS Scientific)
  • 24 × 40–mm coverslips (∼450 mg; Fisher)
  • 100‐ml glass bottle
  • 100‐ml Erlenmeyer flask
  • 45°C water bath
  • Additional reagents and equipment for assessing cell viability (unit 18.8 or appendix 3B)
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 2: In Vivo Chemical Exposure/In Vitro PFC Response to sRBC

  • 20 mg/ml stock S‐9 fraction of pooled female mouse livers (see unit 3.1)
  • 12‐well culture plate
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 3: In Vitro Plaque‐Forming Cell Response TO sRBC with a Metabolic Activation System

  • 10 mg/ml stock solution of LPS from Escherichia coli (strains 0128, 0111, and 055 are commonly used; Difco) or 5 µg/ml DNP‐Ficoll (Biosearch Technologies; http://www.biosearchtech.com/)
  • 50% (v/v) suspension of haptenated sRBC in HBSS, pH 7.0 ( protocol 7; Holsapple, )
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 4: Measuring Plaque‐Forming Cell Response to T‐Independent Antigens

  Materials
  • Sheep as source of blood
  • Alsever's solution (see recipe)
  • Hanks' balanced salt solution, pH 7.0 (see recipe)
  • Isoton (Beckman Coulter)
  • 15‐ or 50‐ml conical polypropylene centrifuge tubes
  • Tabletop centrifuge
  • Accuvette II vials (Beckman Coulter)
  • Coulter Counter (Beckman Coulter)

Support Protocol 1: Preparation of sRBC for Immunization

  Materials
  • Sheep as source of blood
  • Alsever's solution (see recipe)
  • Hank's balanced salt solution (HBSS), pH 7.0 (see recipe)
  • Cacodylate buffer: dissolve 44.8 g cacodylic acid in 1 liter distilled H 2O and adjust pH to 6.9 with HCl
  • 0.015 M picryl sulfonic acid in cacodylate buffer
  • 5.85 mM glycylglycine in cacodylate buffer
  • 50‐ml conical polypropylene centrifuge tubes
  • Refrigerated centrifuge
  • Parafilm
  • Platform rocker
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Figures

Videos

Literature Cited

Literature Cited
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