Measuring Intravascular Migration of Mouse Ly6Clow Monocytes In Vivo Using Intravital Microscopy

L.M. Carlin1, C. Auffray1, F. Geissmann2

1 These authors contributed equally to this work, 2 INSERM U838, Institut Necker, Paris Descartes University, Paris, France
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.33
DOI:  10.1002/0471142735.im1433s101
Online Posting Date:  April, 2013
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This unit describes methods for intravital imaging of monocytes in the vasculature of the dermis and the mesentery in vivo using fluorescent reporter mice, fluorescent dyes, and antibodies. Cx3cr1gfp/gfp (or +), Rag2−/−, Il2rg−/− mice expressing eGFP at the locus of the Cx3cr1 gene, on the Rag2−/− Il2rg−/− C57Bl/6 background, are used. Although aimed at specifically tracking Ly6Clow monocytes, these protocols could readily be adapted to investigate the interaction of other blood leukocytes with the vascular endothelium by use of other fluorescent reporter mice and fluorescently labeled antibodies. Curr. Protoc. Immunol. 101:14.33.1‐14.33.16. © 2013 by John Wiley & Sons, Inc.

Keywords: Ly6Clow monocytes; intravital microscopy; cell tracking; confocal microscopy; blood vessels; in vivo

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Table of Contents

  • Introduction
  • Basic Protocol 1: Intravital Microscopy: Imaging Monocytes in the Dermal Blood Vessels of the Mouse Ear
  • Basic Protocol 2: Intravital Microscopy: Imaging Monocytes in the Mesenteric Blood Vessels of the Mouse
  • Basic Protocol 3: Image Analysis
  • Support Protocol 1: Adoptive Transfer of Monocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Intravital Microscopy: Imaging Monocytes in the Dermal Blood Vessels of the Mouse Ear

  • Cx3cr1gfp/gfp (or +), Rag2−/−, Il2rg−/− mice: mice expressing eGFP at the locus of the Cx3cr1 gene, on the Rag2−/− Il2rg−/− C57Bl/6 background, Cx3cr1gfp mice have been described (Jung et al., ) and are commercially available (The Jackson Laboratory)
  • Ketamine
  • Xylazine
  • Acepromazine
  • Isoflurane
  • Medical oxygen
  • Silicone grease
  • Fluorescent 70‐kDa dextran (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A), Ringer's solution, or other suitable isotonic solution
  • Inverted confocal microscope equipped with 10× (0.4 NA) and 20× (0.5 NA) dry objectives and a thermostat‐controlled chamber maintained at 33°C (also see Smith, )
  • Well plate holder stage insert (sized to clamp a standard footprint 96‐well plate).
  • Tissue culture multi‐well plate lid with circular 2.5‐cm diameter hole punched out, center width‐wise and about 2/3rd along the length, or a custom‐made aluminum plate with the same characteristics—these should be machined to allow free passage of inverted microscope objectives. (i.e., the lower edge of the cutout should be beveled)
  • Oxygen mask suitable for a mouse: a suitable mask can be made by cutting a 5‐ml syringe diagonally (see Fig. )
  • Isoflurane vaporizer and delivery system (if available)
  • Round and square glass coverslips (no 1.5, round 50‐mm diameter, square 22 × 22 mm)
  • Heating pad or lamp
  • Small cotton swabs (e.g., Q‐Tips)
  • Transparent adhesive tape
  • 300‐µl fixed‐needle (30‐G) insulin syringe (U‐100; Becton Dickinson)
  • Additional materials and equipment for injection (unit 1.6), anesthesia (unit 1.4), and euthanasia (unit 1.8) of mice

Basic Protocol 2: Intravital Microscopy: Imaging Monocytes in the Mesenteric Blood Vessels of the Mouse

  • 70% ethanol
  • Surgical tools:
    • Small (∼5‐cm length) straight and curved scissors
    • Straight, curved, and smoothed forceps e.g., suture‐tying forceps
  • Additional reagents and equipment for imaging monocytes in the dermal blood vessels of the mouse ear ( protocol 1)

Basic Protocol 3: Image Analysis

  • Reasonably high‐specification computer (recent high‐performance CPU, ≥8 GB RAM, fast HDD/SSD, fast “gaming”‐style graphics card) with large display
  • Image analysis software: e.g., Imaris (Bitplane), Image J (NIH; see Internet Resources), or Metamorph (Molecular Devices)
  • Spreadsheet/graphing software: e.g., Microsoft Excel, GraphPad Prism

Support Protocol 1: Adoptive Transfer of Monocytes

  • Wild‐type C57Bl/6, CD45.1/.1, or CD45.1/.2 or Cx3cr1gfp/gfp (or +) or Cx3cr1gfp/gfp (or +) Rag2−/−, Il2rg−/− mice
  • Ketamine
  • Xylazine
  • Acepromazine
  • Isoflurane
  • Medical oxygen
  • 100 mM EDTA (Sigma)
  • RBC lysis buffer (see recipe)
  • PBS‐BSA: PBS (without Ca2+/Mg2+; Invitrogen/Life Technologies plus 0.5% BSA (Sigma), filter sterilized
  • RBC lysis buffer (see recipe)
  • Collagenase D
  • DNase I
  • Dulbecco's PBS (with Ca2+/Mg2+; Invitrogen/Life Technologies, cat. no. 14040‐117)
  • 100 mM EDTA stock
  • RPMI/10% FBS (see recipe)
  • Purified anti FcgRII/III clone 2.4G2 (CD32/16; BD Biosciences; Fc blocking reagent)
  • Biotin clone 145‐2C11 (CD3; BD Biosciences)
  • Biotin clone 1D3 (CD19; BD Biosciences)
  • Biotin clone PK136 (NK1.1; BD Biosciences)
  • PE clone AFS98 (CD115; eBiosciences)
  • PE‐Cy7 clone M1/70 (CD11b; BD Biosciences)
  • APC clone RB6‐8C5 (Gr1; BD Biosciences)
  • Streptavidin–Pacific Blue (BD Biosciences)
  • CD45.1/.1 recipient mice
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 1‐ml syringes and 26‐G needles
  • 70‐ and 100‐µm nylon mesh sieves (BD)
  • Refrigerated centrifuge
  • 96‐well U‐bottom tissue culture plates
  • 300‐µl fixed‐needle (30‐G) insulin syringe U‐100; Becton Dickinson
  • Mouse restraining device
  • Additional materials and equipment for blood collection using cardiac puncture (unit 1.7), euthanasia of mice (unit 1.8), preparation of spleen samples (unit 3.1), and counting cells ( appendix 3A)
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Internet Resource
  Rasband, W.S. 1997‐2009. ImageJ. U.S. National Institutes of Health, Bethesda, Maryland, U.S.A.
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