Measurement of Macrophage Growth and Differentiation

Violeta Chitu1, Yee‐Guide Yeung1, Wenfeng Yu1, Sayan Nandi1, E. Richard Stanley1

1 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.20
DOI:  10.1002/0471142735.im1420s92
Online Posting Date:  February, 2011
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Abstract

This unit provides protocols for measuring the abundance and growth of macrophage precursors in agar cultures and the proliferation of isolated mature macrophages in vitro, by either direct cell counting or by DNA measurement. Methods for the immunohistochemical identification of macrophages and the determination of their proliferative status in vivo by immunofluorescence are also included. It also describes methods for characterization of macrophage differentiation through the immunofluorescence analysis of cell‐surface expression of CSF‐1 receptor. Curr. Protoc. Immunol. 92:14.20.1‐14.20.26. © 2011 by John Wiley & Sons, Inc.

Keywords: macrophages; microglia; myeloid; differentiation; CFU‐M assay; macrophage surface markers; CSF‐1 receptor

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of Macrophage Precursor Numbers and Growth In Vitro
  • Basic Protocol 2: Measurement of Macrophage Proliferation In Vitro
  • Basic Protocol 3: Measurement of Macrophage Proliferation In Vitro Using 48‐Well Plates
  • Support Protocol 1: Construction of Calibration Curve for the Conversion of Fluorescence Measurement to Cell Number
  • Basic Protocol 4: Determination of Macrophage Numbers In Vivo
  • Basic Protocol 5: Determination of Microglial Proliferation In Vivo
  • Basic Protocol 6: Measurement of Cell‐Surface CSF‐1R with Biotinylated AFS98 for Characterization of Macrophage Differentiation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Macrophage Precursor Numbers and Growth In Vitro

  Materials
  • Bacto‐Agar (BD Difco)
  • 2× Complete α‐MEM (see recipe)
  • Human recombinant CSF‐1 (Pro‐Spec, http://www.prospecbio.com/)
  • 1× α‐MEM (see recipe)
  • Mice, 6‐10 weeks old
  • 6% (v/v) acetic acid in 1× α‐MEM
  • 4% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS)
  • 41°C water bath
  • 35‐ and 150‐mm diameter tissue culture plates (BD Bioscience)
  • 40‐mesh sieve
  • 100‐mm Petri dishes
  • 5‐ml syringes
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 22‐G needles
  • Inverted microscope equipped with a digital camera
  • ImageJ software (http://rsbweb.nih.gov/ij/download.html)
  • Additional reagents and equipment for mouse euthanasia (unit 1.8), removing spleens and femurs from mice (units 1.9& 6.4, respectively), and counting viable cells using trypan blue dye exclusion ( 3.NaN)

Basic Protocol 2: Measurement of Macrophage Proliferation In Vitro

  Materials
  • Mice for preparing primary macrophages (see protocol 1) or macrophage cell lines
  • Complete medium: 1× α‐MEM (see recipe) supplemented with 10% fetal bovine serum (FBS) and 120 ng/ml human recombinant CSF‐1 (Pro‐Spec, http://www.prospecbio.com/)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold
  • 0.005% (w/v) Zwittergent 3‐14 (Calbiochem) working solution in PBS, freshly prepared from 1% (w/v) stock in H 2O (store stock at 4°C) and kept on ice
  • Coulter Isoton counting fluid
  • 35‐mm diameter tissue culture dishes (BD Bioscience)
  • Inverted microscope
  • Coulter Counter
  • Computer running Microsoft Excel
  • Additional reagents and equipment for preparing primary macrophages from mice ( protocol 1) and counting viable cells using trypan blue dye exclusion ( appendix 3B)

Basic Protocol 3: Measurement of Macrophage Proliferation In Vitro Using 48‐Well Plates

  Materials
  • Macrophage proliferation medium (see recipe)
  • Cells: primary macrophages (see discussions of protocol 2 in the Commentary) or CSF‐1‐dependent macrophage cell lines (e.g. BAC1.2F5, Morgan et al., )
  • 1 µg/ml 4′,6‐diamidino‐2‐phenylindole, dihydrochloride (DAPI); prepare fresh by diluting 1 mg/ml (1000×) stock solution (prepared in double‐distilled water; store in aliquots at –20°C) with staining buffer (see recipe for buffer)
  • 48‐well tissue culture plates
  • Fluorescence microplate reader with filters within ±5 nm of 358 nm for excitation and 461 nm for emission (PolarStar Optima, BMG Labtech) http://www.bmglabtech.com/
  • 3‐ml syringes and 26‐G needles
  • Additional reagents and equipment for counting cells ( appendix 3A& appendix 3B)
CAUTION: DAPI is a known mutagen and should be handled with care. The dye must be disposed of safely and in accordance with applicable local regulations.NOTE: Excitation maximum for DAPI bound to dsDNA is 358 nm, and the emission maximum is 461 nm.

Support Protocol 1: Construction of Calibration Curve for the Conversion of Fluorescence Measurement to Cell Number

  Materials
  • Cells: primary macrophages (see discussions of protocol 2 in the Commentary) or CSF‐1‐dependent macrophage cell lines (e.g. BAC1.2F5, Morgan et al., )
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Macrophage proliferation medium (see recipe)
  • Cell scrapers
  • 15‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Centrifuge
  • 1‐ml syringes and 26‐G needles
  • Additional reagents and equipment for counting cells ( appendix 3A& appendix 3B) and measurement of cell density of nonadherent cells ( protocol 3)

Basic Protocol 4: Determination of Macrophage Numbers In Vivo

  Materials
  • Mouse paraffin‐embedded tissue sections (unit 21.4), 4‐µm thick, on slides
  • Histoclear (National Diagnostics)
  • 90%, 80%, and 70% ethanol (v/v) in double‐distilled water
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Peroxidase quenching solution: 1% (v/v) hydrogen peroxide in 50% methanol/50% PBS; prepare fresh by diluting 30% hydrogen peroxide
  • Primary antibody 1% (v/v) rat anti‐F4/80 antiserum in PBS with 0.05% Tween‐20; 100 µl /slide
  • 0.05% Tween 20 in PBS (see appendix 2A for PBS)
  • Vectastain Peroxidase Rat IgG ABC kit (Vector Laboratories) including:
    • Blocking solution: 2% normal rabbit serum in PBS
    • Biotinylated secondary antibody
    • Reagent A
    • Reagent B
  • 3, 3′ diaminobenzidine (DAB) substrate kit for peroxidase (Vector Laboratories)
  • Harris Hematoxylin solution, modified, 7.5% (Sigma)
  • Bluing solution: ammonia water (0.5% v/v ammonium hydroxide, pH 8) or Scott's Tap Water Substitute for Histology (1% MgSO 4/0.067% NaHCO 3 in tap water)
  • Permount mounting medium (Fisher Scientific)
  • Slide holders and staining trays
  • Humidified chamber for slide incubation: e.g., Tupperware box containing moist paper towels
  • Glass coverslips 24 × 60 mm (Corning)
  • Light microscope
  • Additional reagents and equipment for immunohistochemical staining (unit 21.4)
NOTE: The F4/80 antigen is labile. Mice should be anesthetized and perfused with periodate‐lysine‐paraformaldehyde‐glutaraldehyde (PLPG) fixative (Cecchini et al., ) before dissection. Alternatively, immediately after being euthanized, mice can be injected with 5 ml PLPG in the left ventricle of the heart; however, this procedure may disrupt tissue architecture by breaking capillaries and other small blood vessels.NOTE: All procedures described below are performed at room temperature.

Basic Protocol 5: Determination of Microglial Proliferation In Vivo

  Materials
  • Mice
  • 20 mg/ml BrdU in 154 mM NaCl/7 mM NaOH
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 4% paraformaldehyde (PFA) in PBS, pH 7.4 (see appendix 2A for PBS)
  • 15% (w/v) and 30% (w/v) sucrose in PBS (see appendix 2A for PBS)
  • Embedding medium: Tissue‐Tek Cryo‐Optimal Cutting Temperature (O.C.T.) Compound (Fisher)
  • 2 N HCl
  • 0.1 M sodium borate, pH 8.5
  • 0.1% (w/v) sodium borohydride
  • Blocking solution (see recipe)
  • Primary antibodies: mouse IgG 1 anti‐BrdU (Novo Castra, http://www.leica‐microsystems.com/products/histology‐systems/novocastra‐reagents/) and rabbit IgG anti‐Iba1 (Wako Pure Chemical Industries, Ltd.)
  • Staining buffer: 0.1% (w/v) BSA/0.05% (w/v)saponin in PBS (see appendix 2A for PBS)
  • Secondary antibodies: goat‐anti mouse IgG 1– FITC (1:200) and goat anti‐rabbit IgG‐ TRITC (1:200) (Southern Biotechnology Associates)
  • Antifade‐mounting medium with DAPI (Molecular Probes)
  • Dissecting equipment
  • Rotating shaker
  • Cryostat (see unit 21.4)
  • Embedding molds (see unit 21.4)
  • Staining dishes
  • Humidified dark chamber: opaque plastic box containing moistened paper towels
  • Glass coverslips, 24 × 60 mm (Corning)
  • Fluorescence microscope
  • Additional reagents and equipment for injection of mice (unit 1.6), euthanasia of mice (unit 1.8), and immunohistochemistry (unit 21.4)

Basic Protocol 6: Measurement of Cell‐Surface CSF‐1R with Biotinylated AFS98 for Characterization of Macrophage Differentiation

  Materials
  • Monocytes/macrophages (see Background Information, discussion of protocol 2)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold
  • Staining buffer: 1% (w/v) BSA (Sigma) in PBS (see appendix 2A for PBS), ice cold. (prepare fresh and filter sterilize through a 0.45 µm filter)
  • Mouse BD Fc blocking antibody (rat IgG 2b anti‐mouse CD16/CD32; eBioscience)
  • Biotinylated Rat IgG 2a anti‐CSF‐1R AFS98 (eBioscience)
  • Biotinylated Rat IgG2a Isotype Control (eBioscience)
  • Streptavidin‐PE (BD Pharmingen)
  • 0.01% (w/v) paraformaldehyde/1% NaN 3 in PBS pH 7.4 (see appendix 2A for PBS; optional)
  • 1% (w/v) NaN 3 in PBS, pH 7.4 (see appendix 2A for PBS; optional)
  • Centrifuge
  • 100‐µm mesh filter (optional)
  • Additional reagents and equipment for flow cytometry (Chapter 5)
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Figures

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Literature Cited

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