Evaluation of Toxicity in Mouse Bone Marrow Progenitor Cells

Peace C. Ezeh1, Huan Xu2, Shu Chun Wang2, Sebastian Medina2, Scott W. Burchiel2

1 Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona, 2 College of Pharmacy, Department of Pharmaceutical Sciences, University of New Mexico, Albuquerque, New Mexico
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.9
DOI:  10.1002/0471140856.tx1809s67
Online Posting Date:  February, 2016
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Development of blood cells through hematopoiesis occurs in the bone marrow (BM), and can be adversely impacted by various substances and/or conditions ranging from known therapeutic, intentionally administered xenobiotics to unintentional food additives and exposure to environmental chemicals. The principles underlying the techniques for evaluating toxicity to BM progenitors (erythroid, myeloid, and lymphoid) exploit changes in the normal hematopoietic process, biochemical cell surface and intracellular markers, as well as components of the BM microenvironment. Toxicological investigations following in vivo exposures of mice or in vitro exposures of mouse primary BM cell cultures allow the assessment of the developmental and functional integrity of BM cells, cell population shifts, and adverse biochemical effects due to toxicity. Colony forming unit (CFU) assays and flow cytometry are indispensable techniques in these toxicity studies. © 2016 by John Wiley & Sons, Inc.

Keywords: bone marrow toxicology; hematopoiesis; progenitor cell differentiation; CFU assays; surface and intracellular staining

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Table of Contents

  • Introduction
  • Basic Protocol 1: Mouse Femur Harvest and Bone Marrow Cell Isolation
  • Basic Protocol 2: Assessment of Bone Marrow‐Derived Lymphoid and Myeloid Progenitor Cell Activity In Vitro
  • Basic Protocol 3: Mouse Bone Marrow Cell Surface Markers and Intracellular Staining by Flow Cytometry
  • Reagents And Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Mouse Femur Harvest and Bone Marrow Cell Isolation

  • C57BL/6 J mice, 12 to 16 weeks old (The Jackson Laboratory)
  • 70% (v/v) ethanol
  • 1× Hanks Balanced Salt Solution (HBSS; appendix 2A)
  • Ice
  • Isocove's Modified Dulbecco's Medium (IMDM) with 2% heat‐inactivated FBS (see recipe)
  • Acridine orange/propidium iodide (or other appropriate stain, e.g., trypan blue)
  • CO 2 euthanasia chamber
  • Animal scale
  • Dissection board
  • Sterile surgical instruments including:
  • Scissors
  • Scalpels
  • Forceps
  • Push pins
  • 15‐ml tubes
  • 60 × 15—mm petri dishes, sterile
  • 1‐ml syringe equipped with 25‐G × 15.8‐mm needle
  • 9‐in. Pasteur pipets
  • Automated or manual cell counter
  • Centrifuge

Basic Protocol 2: Assessment of Bone Marrow‐Derived Lymphoid and Myeloid Progenitor Cell Activity In Vitro

  • MethoCult GF M3534 (Stem Cell Technologies) for CFU‐GM assay
  • Mouse methylcellulose complete media (R&D Systems) for CFU‐B assay
  • MethoCult M3334 (Stem Cell Technologies) for CFU‐E assay
  • Sterile water
  • 16‐ml (17 × 100‐mm)‐tubes, sterile
  • Vortex mixer
  • 35‐mm sterile petri dishes
  • Treated 35‐mm cell culture dishes (Stem Cell Technologies)
  • 3‐ml syringes with 16‐G × 3.8‐cm Monoject aluminum hub blunt cannula needle
  • 100‐mm petri dishes
  • 37°C, 5% CO 2 humidified incubator
  • Automated or manual cell counter

Basic Protocol 3: Mouse Bone Marrow Cell Surface Markers and Intracellular Staining by Flow Cytometry

  • Fluorochrome‐conjugated antibodies
  • Bone marrow cells
  • Wash buffer: Dulbecco's phosphate‐buffered saline (DPBS) w/o Ca+2 and Mg+2 containing 1% FBS and 0.9% sodium azide
  • Ammonium chloride lysing solution (1.5 M ammonium chloride, 100 mM sodium bicarbonate, 10 mM disodium EDTA, pH 7.4)
  • 4% paraformaldehyde (see recipe) or cell fixative manufactured solutions (e.g., IC Fixation buffer from eBiosciences)
  • 90% methanol at −20°C
  • Isotope control
  • Centrifuge
  • Flow cytometer
  • Flow tubes
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Literature Cited

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Internet Resources
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