Hematology Testing in Mice

Pontus Lundberg1, Radek Skoda1

1 Department of Biomedicine, Experimental Hematology, University Hospital Basel, Basel, Switzerland
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110088
Online Posting Date:  September, 2011
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Abstract

The mouse is an increasingly important system for the study of both normal and aberrant hematopoiesis. As a model organism, the mouse recapitulates much of human hematopoiesis; however, there are some important differences. Here, the basic approaches for analyzing hematopoiesis in mice are described. In particular, methods are provided for the collection and analysis of peripheral blood, flow cytometry analysis of peripheral blood, bone marrow, and spleen cells, and isolation and transplantation of bone marrow stem cells. Curr. Protoc. Mouse Biol. 1:323‐346 © 2011 by John Wiley & Sons, Inc.

Keywords: mouse hematology; peripheral blood; bone marrow transplantation; hematopoiesis; hematopoietic; stem cell

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Blood Collection in the Mouse via Tail Vein Bleeding
  • Basic Protocol 2: Blood Smears
  • Basic Protocol 3: Flow Cytometry Analysis and Fluorescence Activated Cell Sorting (FACS) of Peripheral Blood
  • Basic Protocol 4: Cardiac Puncture
  • Basic Protocol 5: Isolation and Transplantation of Bone Marrow Cells
  • Support Protocol 1: Flow Cytometry Analysis/Sorting of Bone Marrow Stem/Progenitor Cells
  • Support Protocol 2: Flow Cytometry Analysis of Erythroid Maturation
  • Support Protocol 3: Plating Bone Marrow in Methylcellulose for Colony Assays
  • Support Protocol 4: Serial Replating
  • Basic Protocol 6: Isolation of Spleen Cells
  • Basic Protocol 7: Sublethal/Lethal Irradiation of Mice
  • Basic Protocol 8: Tail Vein Injection
  • Support Protocol 5: Measuring Half Life of Peripheral Blood Cells
  • Support Protocol 6: Isolation of Fetal Mouse Livers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Blood Collection in the Mouse via Tail Vein Bleeding

  Materials
  • Mouse subject
  • 0.9% NaCl
  • BD Microtainer blood collection tube (Becton Dickinson): EDTA for CBC analysis or heparin for blood chemistry
  • Heat lamp
  • Mouse restrainer
  • Scalpel

Basic Protocol 2: Blood Smears

  Materials
  • Peripheral blood ( protocol 1; in some cases the source might be cardiac puncture)
  • Microscope slides (26 × 76 mm)
  • Wright‐Giemsa stain (e.g., Hemacolor, Merck)

Basic Protocol 3: Flow Cytometry Analysis and Fluorescence Activated Cell Sorting (FACS) of Peripheral Blood

  Materials
  • Peripheral blood ( protocol 1)
  • Antibodies (eBioscience, Biolegend, BD Pharmingen)
  • FACS buffer: phosphate‐buffered saline (PBS) containing 0.1% (v/v) fetal bovine serum (FBS) and (optionally) 0.1% (w/v) sodium azide if the cells are not to be cultured/transplanted after sorting
  • Red blood cell lysis buffer (e.g., BD FACS lysis buffer; Becton Dickinson)
  • FACS tubes (Becton Dickinson)
  • Centrifuge
  • Fluorescence‐activated cell sorter (FACS; Robinson et al., )
NOTE: After drawing blood from the mouse, keep the blood in a tube supplemented with EDTA to avoid clotting.

Basic Protocol 4: Cardiac Puncture

  Materials
  • Mouse subject
  • EDTA tubes (BD Microtainer, Becton Dickinson)
  • 1‐ml syringes and 25‐G, 16‐mm needles
  • Additional reagents and equipment for euthanizing mice by CO 2 asphyxiation (Donovan and Brown, )

Basic Protocol 5: Isolation and Transplantation of Bone Marrow Cells

  Materials
  • Mouse subject
  • Phosphate‐buffered saline (PBS), ice cold
  • RPMI medium (e.g., Invitrogen) supplemented with 10% fetal bovine serum (FBS)
  • Red blood cell (RBC) lysis buffer (see recipe)
  • Mouse hematopoietic cell depletion kit (R&D Systems)
  • Dissecting equipment including forceps and scissors
  • Mortar and pestle
  • Cell strainers (e.g., Becton Dickinson)
  • 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Additional reagents and equipment for euthanizing mice by CO 2 asphyxiation (Donovan and Brown, )

Support Protocol 1: Flow Cytometry Analysis/Sorting of Bone Marrow Stem/Progenitor Cells

  Materials
  • Bone marrow cells ( protocol 5)
  • Mouse hematopoietic cell depletion kit (R&D Systems)
  • FACS buffer: phosphate‐buffered saline (PBS) containing 2% (v/v) fetal bovine serum (FBS) and (optionally) 0.1% (w/v) sodium azide if the cells are not to be cultured/transplanted after sorting
  • Antibody staining cocktail
  • Centrifuge
  • Flow cytometer/cell sorter supporting multi‐color analysis (see Robinson et al., ) and FACS tubes

Support Protocol 2: Flow Cytometry Analysis of Erythroid Maturation

  Materials
  • Bone marrow cells ( protocol 5) or spleen cells ( protocol 10)
  • FACS buffer: phosphate‐buffered saline (PBS) containing 0.1% (v/v) fetal bovine serum (FBS) and (optionally) 0.1% (w/v) sodium azide if the cells are not to be cultured/transplanted after sorting
  • Antibody staining cocktail
  • Flow cytometer/cell sorter supporting multi‐color analysis (see Robinson et al., ) and FACS tubes

Support Protocol 3: Plating Bone Marrow in Methylcellulose for Colony Assays

  Materials
  • MethoCult methylcellulose medium containing the appropriate cytokine combination (StemCell Technologies)
  • Bone marrow cells ( protocol 5)
  • IMDM medium (e.g., Invitrogen) supplemented with 2% fetal bovine serum (FBS)
  • 16‐G needle
  • 3‐ml syringe
  • 35‐mm and 150‐mm Petri dishes (e.g., Becton Dickinson)
  • 37°C, 5% CO 2 humidified incubator
  • Additional reagents and equipment for red blood cell lysis (see protocol 5)

Support Protocol 4: Serial Replating

  Materials
  • MethoCult methylcellulose medium containing the appropriate cytokine combination (StemCell Technologies)
  • Petri dishes containing cells from first methylcellulose plating ( protocol 8)
  • RPMI medium (e.g., Invitrogen) supplemented with 10% fetal bovine serum (FBS)
  • 15‐ml conical tubes
  • Centrifuge
  • Additional reagents and equipment for plating bone marrow cells in methylcellulose medium ( protocol 8) and counting viable cells by trypan blue exclusion (e.g., Sandell and Sakai, )

Basic Protocol 6: Isolation of Spleen Cells

  Materials
  • Mouse subject
  • Phosphate‐buffered saline (PBS)
  • Dissecting instruments
  • 40‐µm mesh cell strainers (Becton Dickinson)
  • 6‐well tissue culture plates
  • 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Additional reagents and equipment for euthanasia of the mouse by CO 2 asphyxiation (Donovan and Brown, )

Basic Protocol 7: Sublethal/Lethal Irradiation of Mice

  Materials
  • Mouse subject, 8 to 10 weeks old
  • Medicated water (treated with Napil, available from various drug companies)
  • γ‐irradiation source (Cs137 mouse irradiator; Theratronics, http://www.theratronics.ca/)
  • Irradiation‐compatible box (Theratronics, http://www.theratronics.ca/)

Basic Protocol 8: Tail Vein Injection

  Materials
  • Mouse subject (lethally irradiated; protocol 11)
  • Cells: e.g., bone marrow cell suspension ( protocol 5)
  • 70% ethanol
  • PBS
  • Heat lamp
  • Mouse restrainer
  • Insulin syringe with 29‐G needle (micro‐fine, 0.5 ml, Becton Dickinson)

Support Protocol 5: Measuring Half Life of Peripheral Blood Cells

  Materials
  • Mouse subject
  • 3 µg/µl NHS‐biotin (Sigma) in 0.9% NaCl
  • Phosphate‐buffered saline (PBS)
  • Insulin syringe with 29‐G needle
  • Heat lamp
  • Mouse restrainer
  • FACS tubes (e.g., BD)
  • Additional reagents and equipment for tail vein injection ( protocol 12) and tail vein bleeding ( protocol 1)

Support Protocol 6: Isolation of Fetal Mouse Livers

  Materials
  • Mice
  • Phosphate‐buffered saline (PBS)
  • RPMI medium (e.g., Invitrogen) supplemented with 10% fetal bovine serum (FBS)
  • Dissecting instruments
  • Petri dishes (e.g., Becton Dickinson)
  • Cell strainers (e.g., BD)
  • Additional reagents and equipment for mating of mice (Ayadi et al., ), euthanasia of mice (Donovan and Brown, ), and isolation of cells (as for spleen cells; protocol 14)
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Figures

Videos

Literature Cited

Literature Cited
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