Choosing and Preparing Antigen‐Presenting Cells

Clifford V. Harding1, David Canaday1, Lakshmi Ramachandra1

1 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 16.1
DOI:  10.1002/0471142735.im1601s88
Online Posting Date:  February, 2010
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Abstract

The first issue in selecting a system for antigen‐presentation experiments is to define the appropriate type of antigen‐presenting cell (APC) to study. For some experiments, crude preparations such as splenocytes or peripheral blood mononuclear cells (PBMCs) may suffice to provide APC function for stimulating T cells. This unit develops approaches for preparation of more defined APC populations, including dendritic cells (DCs), macrophages, and B lymphocytes, the three types of “professional” APC. Each of these cell types exists in different stages of differentiation, maturation, and activation, or in some cases different lineages. For example, dendritic cells may be divided into subsets, including myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). Each APC type has an important antigen‐presentation function, although they contribute to different aspects of the immune response. Therefore, selection of an APC type for study must include consideration of the stage or aspect of immune response that is to be modeled in the experiment. Curr. Protoc. Immunol. 88:16.1.1‐16.1.30. © 2010 by John Wiley & Sons, Inc.

Keywords: antigen‐presenting cells; antigen processing; dendritic cells; macrophages; B lymphocytes; PBMCs; monocytes

     
 
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Table of Contents

  • Introduction
  • Preparation of Murine Dendritic Cells
  • Basic Protocol 1: Preparing Bone Marrow–Derived DCs with Flt‐3 Ligand
  • Basic Protocol 2: Isolating DCs from Spleen
  • Preparation of Human Dendritic Cells
  • Basic Protocol 3: Isolating mDCs from Blood
  • Support Protocol 1: Preparation of Peripheral Blood Mononuclear Cells
  • Support Protocol 2: Freezing PBMCs, Monocytes, or B Cells
  • Basic Protocol 4: Preparing Monocyte‐Derived DCs
  • Support Protocol 3: Isolating Human Monocytes
  • Murine Macrophages
  • Basic Protocol 5: Preparing Bone‐Marrow‐Derived Macrophages
  • Support Protocol 4: Preparing LADMAC Conditioned Medium
  • Support Protocol 5: Preparing L‐929 Conditioned Medium
  • Basic Protocol 6: Eliciting Peritoneal Macrophages with ConA
  • Alternate Protocol 1: Eliciting Peritoneal Macrophages with Listeria monocytogenes
  • Alternate Protocol 2: Eliciting Peritoneal Macrophages by Sequential Injection of Listeria monocytogenes and Proteose Peptone
  • Support Protocol 6: Preparing Listeria monocytogenes Stocks
  • Basic Protocol 7: Isolating Alveolar Macrophages
  • Support Protocol 7: Preparing 2,2,2‐Tribromoethanol
  • Human Monocytes and Macrophages
  • Basic Protocol 8: Isolating Human Monocytes
  • Basic Protocol 9: Preparing Human Monocyte‐Derived Macrophages
  • Using APC Cell Lines
  • Basic Protocol 10: Example: Human THP‐1 Cells
  • Murine B Cells
  • Basic Protocol 11: Preparing LPS‐Stimulated B Lymphoblasts for Use as Antigen‐Presenting Cells
  • Human B Cells
  • Basic Protocol 12: Isolation of B Cells from Blood
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparing Bone Marrow–Derived DCs with Flt‐3 Ligand

  Materials
  • Mice of appropriate H‐2 haplotype (see Table 97.80.4711A.1C.1)
  • RPMI 1640 medium, serum free (Hyclone)
  • ACK lysing buffer (see recipe; also available from Bio‐Whittaker)
  • Complete RPMI‐10 medium ( appendix 2A), room temperature
  • 0.4% (w/v) trypan blue solution (Sigma; also see appendix 3B)
  • Flt‐3 ligand (Bio X Cell, http://www.bxcell.com/)
  • 5‐ml syringes
  • 25‐G needles
  • 50‐ml conical tube (Falcon)
  • 70‐µm nylon cell strainers (Falcon)
  • Sorvall RT6000 refrigerated tabletop centrifuge and HB‐1000 rotor (or equivalent)
  • 6‐well tissue culture plates
  • 96‐well flat‐ or round‐bottom tissue culture plates
  • Additional reagents and equipment for euthanasia (unit 1.8), dissecting mouse femurs (unit 14.1), and counting viable cells ( appendix 3A& appendix 3B)
NOTE: All solutions, equipment, and tissue culture materials coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. Solutions and materials should be free of endotoxin or other contaminants that may drive DC maturation (take particular care in selecting protein preparations, e.g., bovine serum albumin or antigen preparations, as these are frequently contaminated with endotoxin, which may introduce problems and artifacts).NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Isolating DCs from Spleen

  Materials
  • Mice of appropriate H‐2 haplotype (see Table 97.80.4711A.1C.1)
  • RPMI 1640 medium, serum free (Hyclone)
  • 10× digestion mix: collagenase type III (Worthington) at 1500 U/ml with DNase I (Sigma) at 300 U/ml
  • Complete RPMI‐10 medium ( appendix 2A)
  • ACK lysing buffer (see recipe; also available from Bio‐Whittaker)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Cell isolation buffer: e.g., MACS buffer from Miltenyi Biotec
  • Commercially available kits for magnetic cell separation/isolation (e.g., MACS Cell Separation kits from Miltenyi Biotec or equivalent from StemCell Technologies)
  • 0.4% (w/v) trypan blue solution (Sigma; also see appendix 3B)
  • 100‐mm petri dish (non–tissue culture treated)
  • Scissors, sterile
  • 50‐ml conical tubes (Falcon)
  • Rotary mixer
  • 70‐µm nylon cell strainers (Falcon)
  • Sorvall RT6000 refrigerated tabletop centrifuge and HB‐1000 rotor (or equivalent)
  • 96‐well flat‐bottom tissue culture plates
  • Additional reagents and equipment for euthanasia (unit 1.8), removing the spleen (unit 1.9), preparation of splenocyte suspension (unit 3.7) and counting viable cells ( appendix 3A& appendix 3B)
NOTE: All solutions, equipment, and tissue materials coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. Solutions and materials should be free of endotoxin or other contaminants that may drive DC maturation (take particular care in selecting protein preparations, e.g., bovine serum albumin or antigen preparations, as these are frequently contaminated with endotoxin, which may introduce problems and artifacts).NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Isolating mDCs from Blood

  Materials
  • PBMC (fresh, NOT frozen; protocol 4)
  • Cell isolation buffer: e.g., MACS buffer from Miltenyi Biotec
  • Commercially available kits for magnetic cell separation/isolation of mDCs (e.g., MACS Cell from Miltenyi Biotec or equivalent from StemCell Technologies, Inc.)
  • P2 medium (see recipe)
  • 50‐ml conical tubes (Falcon)
  • 96‐well round‐bottom tissue culture plates
NOTE: All solutions and tissue culture materials coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. Solutions and materials should be free of endotoxin or other contaminants that may drive DC maturation (take particular care in selecting protein preparations, e.g., bovine serum albumin or antigen preparations, as these are frequently contaminated with endotoxin, which may introduce problems and artifacts).

Support Protocol 1: Preparation of Peripheral Blood Mononuclear Cells

  Materials
  • Heparinized blood (blood should be kept rocking until ready to use)
  • Serum‐free RPMI 1640 or DMEM medium (Hyclone), room temperature
  • Ficoll‐Hypaque (Ficoll Paque Plus; GE Healthcare)
  • Complete RPMI‐10 medium ( appendix 2A), P2 medium (see recipe), or X‐Vivo medium (Thermo Fisher); room temperature
  • 3% (v/v) acetic acid
  • 50‐ml conical tubes (Falcon)
  • Sorvall RT6000 refrigerated tabletop centrifuge and HB‐1000 rotor (or equivalent centrifuge with swinging‐bucket rotor)
  • Additional reagents and equipment for cell counting ( appendix 3A& appendix 3B)
NOTE: All solutions and tissue culture materials 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 unless otherwise specified.

Support Protocol 2: Freezing PBMCs, Monocytes, or B Cells

  Materials
  • PBMCs, monocytes, or B cells to be frozen (see appropriate protocols in this unit)
  • Freezing medium: 90% decomplemented fetal bovine serum FBS (Hyclone)/10% DMSO (Sigma); mix FBS and DMSO prior to filter sterilization.
  • Sorvall RT6000 refrigerated tabletop centrifuge and HB‐1000 rotor (or equivalent)
  • Cryogenic vials (Nalgene)
  • Freezing container (Mr. Frosty from Nalgene)
NOTE: All solutions, equipment and tissue culture materials coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

Basic Protocol 4: Preparing Monocyte‐Derived DCs

  Materials
  • PBMCs, fresh or frozen (see protocol 4)
  • Cell isolation buffer: e.g., MACS buffer from Miltenyi Biotec
  • 50‐ml conical tubes (Falcon)
  • Commercially available kits for positive or negative cell separation/isolation of CD14+ human monocytes (e.g., MACS Cell Separation kits from Miltenyi Biotec or immunomagnetic isolation kits from StemCell Technologies)
NOTE: All solutions and tissue culture materials coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. Solutions and materials should be free of endotoxin or other contaminants that may stimulate monocytes (take particular care in selecting protein preparations, e.g., bovine serum albumin or antigen preparations, as these are frequently contaminated with endotoxin, which may introduce problems and artifacts).

Support Protocol 3: Isolating Human Monocytes

  Materials
  • Mice of appropriate H‐2 haplotype (see Table 97.80.4711A.1C.1)
  • Complete DMEM‐10 medium ( appendix 2A)
  • 25% (v/v) LADMAC‐conditioned medium (see protocol 10) in complete DMEM‐10 medium or 20% (v/v) L‐929 conditioned medium (see protocol 14) in complete DMEM‐10 medium ( appendix 2A)
  • 3% (v/v) acetic acid
  • 0.4% (w/v) trypan blue solution (Sigma)
  • 0.05 (w/v) trypsin/0.53 mM EDTA solution (Invitrogen)
  • 100 U/ml (10 ng/ml) recombinant murine interferon γ (IFN‐γ; Genzyme)
  • 5‐ml syringes
  • 25‐G needles
  • 50‐ml conical tubes (Falcon)
  • 10‐ml pipet
  • 70‐µm nylon cell strainers (Falcon)
  • Sorvall RT6000 refrigerated tabletop centrifuge and HB‐1000 rotor (or equivalent)
  • 100‐mm petri dishes (non–tissue culture treated)
  • 96‐well flat‐bottom tissue culture plates
  • Additional reagents and equipment for euthanasia (unit 1.8), dissecting mouse femurs (unit 14.1), and counting viable cells ( appendix 3A& appendix 3B)
NOTE: All solutions, equipment and tissue culture ware 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 unless otherwise specified.

Basic Protocol 5: Preparing Bone‐Marrow‐Derived Macrophages

  Materials
  • Culture of LADMAC cells (ATCC no. CRL‐2420) in 75‐cm2 tissue culture flasks
  • Complete EMEM‐10 medium (see Reagents and Solutions)
  • 75‐cm2 and 175‐cm2 tissue culture flasks
  • 250‐ml conical tubes, sterile (Corning)
  • Sorvall RT6000 refrigerated tabletop centrifuge and HB‐1000 rotor (or equivalent)
  • 0.22‐µm filter
NOTE: All solutions and tissue culture ware 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 unless otherwise specified.

Support Protocol 4: Preparing LADMAC Conditioned Medium

  Materials
  • Adherent culture of L‐929 cells (ATCC no. CCL1) in 25‐cm2 tissue culture flasks
  • Phosphate‐buffered saline (PBS; appendix 2A), room temperature
  • 0.05% (w/v) trypsin/0.53 mM EDTA solution (Invitrogen)
  • Complete DMEM‐10 medium ( appendix 2A)
  • 25‐cm2 tissue culture flasks (and other flasks of appropriate size)
  • Sorvall RT6000 refrigerated tabletop centrifuge and HB‐1000 rotor (or equivalent)
  • 15‐ml centrifuge tubes, sterile
  • 0.45‐µm filter
NOTE: All solutions and tissue culture ware 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 unless otherwise specified.

Support Protocol 5: Preparing L‐929 Conditioned Medium

  Materials
  • Concanavalin A (ConA, type IV, Sigma)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Mice of the appropriate H‐2 haplotype (see Table 97.80.4711A.1C.1)
  • DMEM medium (Hyclone, appendix 2A), ice‐cold
  • 3% (v/v) acetic acid
  • 0.4% (w/v) trypan blue solution (Sigma)
  • Complete DMEM‐10 medium ( appendix 2A)
  • 0.22‐µm filters
  • 23‐G needles
  • 10‐ and 30‐ml syringes
  • 50‐ml centrifuge tubes (Falcon)
  • Sorvall RT6000 refrigerated tabletop centrifuge with HB‐1000 rotor (or equivalent)
  • 96‐well flat‐bottom tissue culture plates
  • Inverted microscope
  • Additional reagents and equipment for i.p. injection of mice (unit 1.6), harvesting peritoneal exudate (unit 14.1), and counting viable cells using a hemacytometer ( appendix 3A& appendix 3B)
NOTE: All solutions, equipment and tissue culture materials 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 unless otherwise specified.

Basic Protocol 6: Eliciting Peritoneal Macrophages with ConA

  • Listeria monocytogenes stock ( protocol 14)

Alternate Protocol 1: Eliciting Peritoneal Macrophages with Listeria monocytogenes

  • 10% (w/v) proteose peptone (BD Difco)

Alternate Protocol 2: Eliciting Peritoneal Macrophages by Sequential Injection of Listeria monocytogenes and Proteose Peptone

  Materials
  • Four naive mice (e.g., CBA/J)
  • Culture of Listeria monocytogenes (ATCC no.15353)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 100‐mm brain/heart infusion agar (BD Difco) plates, sterile
  • Brain heart infusion broth (BD Difco), sterile
  • Bacterial freezing solution: 20% (v/v) glycerol in PBS, filter sterilized and stored at 4°C
  • 70‐µm nylon cell strainers (Falcon)
  • 50‐ml disposable centrifuge tubes, sterile
  • Bacterial cell spreader, e.g., L‐shaped glass rod, flame sterilized
  • Plating turntable (optional)
  • Inoculating loop, sterile
  • Spectrophotometer and cuvettes
  • High‐speed centrifuge bottles with caps, sterile
  • Sample rotator
  • Sorvall RC‐5 centrifuge with GSA rotor (or equivalent)
  • 1‐ml cryovials (Nalgene)
  • Additional reagents and equipment for intraperitoneal injection (unit 1.6), euthanasia (unit 1.8), removing the spleen (unit 1.9), and recovery of spleen cells (unit 3.1)
NOTE: All solutions, equipment and tissue culture materials coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. Listeria monocytogenes is a Biohazard Level 2 human pathogen, and appropriate precautions and decontamination should be performed. Pregnant women and immunocompromised individuals should not work with L. monocytogenes.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 6: Preparing Listeria monocytogenes Stocks

  Materials
  • Mice of appropriate H‐2 haplotype (see Table 97.80.4711A.1C.1)
  • Tribromoethanol for anesthetizing mice ( protocol 16)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Complete RPMI‐10 ( appendix 2A)
  • 0.4% (w/v) trypan blue solution (Sigma)
  • 20 U/ml (2 ng/ml) recombinant murine IFN‐γ (Genzyme)
  • 1‐ml syringes
  • 27‐G needle
  • Styrofoam board and dissecting pins
  • Dissecting instruments
  • 18‐G angiocatheter (BD Biosciences)
  • Sorvall RT6000 refrigerated tabletop centrifuge with HB‐1000 rotor (or equivalent)
  • 96‐well flat‐bottom tissue culture plates
  • Additional reagents and equipment for intraperitoneal injection (unit 1.6), dissecting mouse femurs (unit 14.1), and counting viable cells ( appendix 3A& appendix 3B)
NOTE: All solutions, equipment and tissue culture materials 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 unless otherwise specified.

Basic Protocol 7: Isolating Alveolar Macrophages

  Materials
  • 2‐methyl‐2‐butanol (t‐amyl alcohol, Sigma)
  • 2,2,2‐tribromoethanol (Sigma)
  • 50°C water bath
  • 0.22‐µm Stericup filters (Millipore)
  • Polystyrene tubes

Support Protocol 7: Preparing 2,2,2‐Tribromoethanol

  Materials
  • Monocytes (fresh or frozen; see other protocols in unit)
  • Cell isolation buffer: e.g., MACS buffer from Miltenyi Biotec
  • P2 medium (see recipe)
  • Recombinant human macrophage‐colony stimulating factor (M‐CSF, R&D Systems)
  • 0.05 (w/v) trypsin/0.53 mM EDTA solution (Life Technologies)
  • 50‐ml conical tubes (Falcon)
  • Sorvall RT6000 refrigerated tabletop centrifuge with HB‐1000 rotor (or equivalent)
  • 96‐well or 6‐well plates (or other appropriate‐sized plates)
NOTE: All solutions and tissue culture ware 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 unless otherwise specified.

Basic Protocol 8: Isolating Human Monocytes

  Materials
  • THP‐1 cells (ATCC no. TIB‐202)
  • Complete RPMI‐10 ( appendix 2A)
  • Phorbol myristate acetate (PMA, Sigma)
  • Recombinant human IFN‐γ (Genzyme)
  • 96‐well flat‐bottom tissue culture plates (or other appropriate‐sized plates)
NOTE: All solutions and tissue culture materials 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 unless otherwise specified.

Basic Protocol 9: Preparing Human Monocyte‐Derived Macrophages

  Materials
  • Suspension of splenocytes (unit 3.1) in complete DMEM‐10 medium ( appendix 2A)
  • Complete DMEM‐10 medium ( appendix 2A)
  • ACK lysing buffer (see recipe and unit 3.1)
  • Lipopolysaccharide (LPS) from E. coli (e.g., Invivogen)
  • 100‐mm tissue culture dishes
  • 50‐ml conical centrifuge tubes, sterile
  • Sorvall RT6000 tabletop centrifuge and Sorvall HB‐1000 rotor (or equivalent)
  • 75‐cm2 tissue culture flasks
  • Additional reagents and equipment for lysing erythrocytes (unit 3.1), and counting cells ( appendix 3A& appendix 3B)
NOTE: All solutions, equipment and tissue culture materials 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 unless otherwise specified.
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Literature Cited

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