Hematology Testing in Mice

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 CO₂ 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 CO₂ 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% CO₂ 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 CO₂ 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

Figure 1. Tail vein bleeding. (A ) Position the scalpel so that it is touching the artery. (B ) Directly after making the cut, blood should start dripping. (C ) Collect the blood in a tube already containing EDTA until the desired amount is obtained. Do not collect more blood than recommended, as excessive bleeding could harm the mice.

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Figure 2. The process of performing a blood smear. (A ) See , step 1. (B) See , step 2. (C ) See , step 3. (D ) See , step 4.

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Figure 3. Representative scattergrams for peripheral blood. (A ) Scattergram of peripheral blood before RBC lysis in log scale with platelets (1), and red blood cells together with lymphocytes (2). (B ) Scattergram of peripheral blood after RBC lysis: lymphocytes (3), granulocytes (4), and monocytes (5).

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Figure 4. Cardiac puncture. (A ) Insert the needle in an angled fashion. (B ) Retract the plunger very slowly to collect the blood. If the blood stops flowing, rotating the needle or pushing/pulling a bit might restore the blood flow.

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Figure 5. Analysis of erythroid maturation in bone marrow.

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Figure 6. Ventral view of a mouse with the thorax opened. (1) Heart. (2) Spleen.

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Figure 7. Mouse tail vein injection. (A ) Notice the positioning of the mouse, which is lying on its side in the restrainer to expose the tail vein. (B ) After a successful injection a drop of blood should be visible.

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Videos

Literature Cited

Literature Cited
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Hematology Testing in Mice

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited