Measurement of Interleukin 3 and Other Hematopoietic Cytokines, such as GM‐CSF, G‐CSF, M‐CSF, Erythropoietin, Steel Factor, and Flt‐3 Ligand

Scott H. Cooper1, Hal E. Broxmeyer1

1 Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.4
DOI:  10.1002/0471142735.im0604s37
Online Posting Date:  May, 2001
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Abstract

Detection and quantitation of IL‐3 and other hematopoietic cytokines in serum samples and culture supernatants has played an important role in understanding their function and pleiotropic properties during the process of hematopoiesis. Several methods for detecting and quantitating cytokines—such as the colony‐stimulating factors granulocyte/macrophage colony‐stimulating factor (GM‐CSF), granulocyte colony‐stimulating factor (G‐CSF), macrophage colony‐stimulating factor (M‐CSF, also called CSF‐1), interleukin 3 (IL‐3), and erythropoietin (Epo), as well as two potent costimulating cytokines steel factor (SLF) and Flt3/Flk‐2 ligand (Flt3‐L)—are described in this unit.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Hematopoietic Cytokines Using Granulocyte/Macrophage Cell Growth in Semisolid Medium
  • Basic Protocol 2: Detection of Hematopoietic Cytokines Using Erythroid (BFU‐E) and Multipotential (CFU‐GEMM) Cell Growth in Semisolid Medium
  • Support Protocol 1: Preparation of Mouse Bone Marrow Cells
  • Basic Protocol 3: Detection of Hematopoietic Cytokines Using Cytokine‐Dependent Cell Lines
  • Support Protocol 2: Maintenance of Cytokine‐Dependent Cell Lines
  • Support Protocol 3: Production of WEHI‐3 Conditioned Medium
  • Basic Protocol 4: Detection of Cytokines by ELISA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Hematopoietic Cytokines Using Granulocyte/Macrophage Cell Growth in Semisolid Medium

  Materials
  • Primary target cells (see Table 6.4.1 and unit 7.1)
    Table 6.4.1   MaterialsCFU‐GM Assay Mixture

    Component Stock concentration Final concentration Volume/10 ml a
    Agar 0.6% (w/v) 0.3% (w/v) 5 ml
    2 × McCoy 5A 0.75× 3.75 ml
    FBS (heat‐inactivated) b NA 10% (v/v) 1.0 ml
    Cells
    Human bone marrow 2 × 106 cells/ml 5 × 104 cells/ml 0.25 ml
    Human cord blood 1 × 106 cells/ml 2.5 × 104 cells/ml 0.25 ml
    Human peripheral blood leukocytes 8 × 106 cells/ml 2.0 × 105 cells/ml 0.25 ml
    Murine bone marrow 2 × 106 cells/ml 5 × 104 cells/ml 0.25 ml

     aThe 10‐ml volume is given strictly as a guideline for proportions; scaling up to a larger volume is required for larger assays.
     bHeat inactivation of FBS has been found to allow better and more consistent cloning efficiencies for CFU‐GM‐derived colonies.
  • recipe0.6% agar (Bacto agar, Difco) or recipe0.8% agarose (SeaPlaque agarose, FMC Bioproducts), autoclaved
  • Cytokine standard (Genzyme, Amgen, or R & D Systems)
  • recipe1× and 2× McCoy 5A medium (see recipe)
  • Sample to be tested
  • FBS (HyClone), heat inactivated 30 min at 56°C
  • 45°C water bath
  • 10‐ml tubes, sterile
  • 10 × 35–mm petri dishes, sterile
  • Scoring grid: 75 × 50–mm glass slide etched with 5‐ or 6‐mm squares
  • Additional reagents and equipment for Ficoll‐Hypaque gradient centrifugation (unit 7.1) and counting cells ( appendix 3A)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are preferably performed in a humidified 37°C, 5% CO 2, 5% O 2 incubator; however, an ambient O 2 concentration of 21% is acceptable.

Basic Protocol 2: Detection of Hematopoietic Cytokines Using Erythroid (BFU‐E) and Multipotential (CFU‐GEMM) Cell Growth in Semisolid Medium

  Materials
  • Primary target cells (see Table 6.4.2 and unit 7.1)
    Table 6.4.2   MaterialsBFU‐E/CFU‐GEMM Assay Mixture

    Component Stock concentration Final concentration Volume/10 ml c
    Reagents
    Methylcellulose d 2.3% (w/v) 1.0% (w/v) 4.3 ml
    IMDM 2.26 ml
    FBS (not heat inactivated) e NA 30% (v/v) 3.0 ml
    200 mM glutamine 200 mM 2 mM 0.1 ml
    2‐ME 10−2 M 3.3 × 10−5 M 0.033 ml
    Erythropoietin f 200 U/ml 1 U/ml 0.05 ml
    Cells
    Human bone marrow 2 × 106 cells/ml 5 × 104 cells/ml 0.25 ml
    Human cord blood 1 × 106 cells/ml 2.5 × 104 cells/ml 0.25 ml
    Human peripheral blood leukocytes 8 × 106 cells/ml 2.0 × 105 cells/ml 0.25 ml
    Murine bone marrow 2 × 106 cells/ml 5 × 104 cells/ml 0.25 ml

     cThe 10‐ml volume is given strictly as a guideline for proportions.
     dMethylcellulose is purchased prepared in medium; other reagents should be prepared in medium.
     eFBS that has not been heat inactivated provides for better growth and color colonies in this assay.
     fErythropoietin (Epo) alone will result in stimulation of only BFU‐E‐derived erythroid colonies. A final concentration of 100 U/ml interleukin 3 (IL‐3) and/or 50 ng/ml steel factor (SLF) added to 1 U/ml Epo will stimulate CFU‐GEMM colonies. For mouse cells, add a final concentration of 0.1 mM hemin (Eastman Kodak) to enhance the red color for BFU‐E and CFU‐GEMM colonies. Without Epo, no BFU‐E or CFU‐GEMM colonies will form.
  • Iscove modified Dulbecco medium (IMDM) containing 100 U/ml penicillin G and 100 µg/ml streptomycin
  • FBS (HyClone), not heat‐inactivated
  • 200 mM glutamine
  • recipe0.01 M 2‐mercaptoethanol (2‐ME), sterile (see recipe)
  • 200 U/ml erythropoietin (Epogen, Amgen)
  • Cytokine standard (Genzyme, Amgen, or R & D Systems)
  • Sample to be tested
  • Methylcellulose (MethoCult, StemCell Technologies)
  • 10‐ml tube, sterile
  • 10‐ml syringe with blunt‐end 13‐ or 16‐G needle, sterile
  • Scoring grid: 75 × 50–mm glass slide etched with 5‐ or 6‐mm squares
  • Additional reagents and equipment for Ficoll‐Hypaque gradient centrifugation (unit 7.1) and counting cells ( appendix 3A)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2, 5% O 2 incubator; however, an ambient O 2 concentration of 21% is acceptable.

Support Protocol 1: Preparation of Mouse Bone Marrow Cells

  Materials
  • Female B 6D 2F 1 mice, 7 to 10 weeks old
  • 70% ethanol
  • recipeComplete 1× McCoy 5A medium (see recipe)
  • Dissecting equipment: forceps and scissors, sterile
  • 10‐ml syringes with 23‐ and 25‐G needles, sterile
  • 15‐ml conical centrifuge tube, sterile
  • Additional reagents and equipment for counting cells ( appendix 3A)
NOTE: After the animal has been sacrificed, work in a clean environment and maintain sterile technique.

Basic Protocol 3: Detection of Hematopoietic Cytokines Using Cytokine‐Dependent Cell Lines

  Materials
  • recipeComplete RPMI‐10 medium (see recipe)
  • Cytokine standard, human or murine, in recipecomplete RPMI‐10 (e.g., 500 U/ml IL‐3, Genzyme)
  • Cytokine‐dependent cell line: e.g., for IL‐3, FDC‐P2 (murine), M07e (human), or TF‐1 (human; ATCC CRL 2003)
  • Samples to be tested
  • Low‐specific‐activity [3H]thymidine (2 Ci/mmol)
  • 96‐well flat‐bottom microtiter plate (proliferation assays; e.g., Costar) or 10 × 35–mm petri dishes (colony‐forming assays)
  • Scoring grid: 75 × 50–mm glass slide etched with 5‐ or 6‐mm squares
  • Additional reagents and equipment for counting cells ( appendix 3A), determining cell viability ( appendix 3B),and for [3H]thymidine incorporation (unit 3.17), reduction of MTT (unit 6.3), or colony‐formation in semisolid medium (see Basic Protocols protocol 11 or protocol 22)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2, 5% O 2 incubator unless otherwise indicated.

Support Protocol 2: Maintenance of Cytokine‐Dependent Cell Lines

  Materials
  • recipeComplete RPMI‐10 medium (see recipe) or other medium recommended by supplier of cell line
  • WEHI‐3 cells (ATCC #TIB 68)
  • Recombinant human IL‐3 (rhuIL‐3) or GM‐CSF (rhuGM‐CSF; Genzyme, Amgen, or R & D Systems) or other cell maintenance factor (e.g., conditioned medium from cytokine‐producing cells)
  • 150‐cm2 tissue culture flasks
  • 50‐ml centrifuge tubes, sterile
  • 0.2‐µm filter units
  • Additional reagents and equipment for quantitating IL‐3 by proliferation assay (see protocol 4) or ELISA (see protocol 7)
NOTE: All equipment and solutions coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator.

Support Protocol 3: Production of WEHI‐3 Conditioned Medium

  Materials
  • Primary anti‐IL‐3 monoclonal antibody (MAb, e.g., R&D Systems, Dako, or Serotech)
  • recipe0.2 M carbonate buffer, pH 9.4 (see recipe)
  • recipeWash buffer (see recipe)
  • recipeBlocking buffer (see recipe)
  • IL‐3 or other cytokine standards (e.g., R & D Systems)
  • Sample(s) to be tested (e.g., conditioned medium from IL‐3‐producing cells; see protocol 6)
  • Secondary conjugated anti‐IL‐3 monoclonal or polyclonal antibody (e.g., alkaline phosphatase–conjugated anti‐IL‐3, R & D Systems)
  • Enzyme substrate (e.g., 1 mg/ml p‐nitrophenyl phosphate, NPP, Sigma) in appropriate substrate buffer
  • Disposable ELISA plates
  • 500‐ml wash bottle
  • Microtiter plate reader
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Figures

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Literature Cited

Literature Cited
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