CFU‐MK Assay for Acute Thrombocytopenia

Dominique Parent‐Massin1, Yann Sibiril1

1 UFR Sciences, Université de Bretagne Occidentale, Brest, France
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.5
DOI:  10.1002/0471140856.tx2005s37
Online Posting Date:  August, 2008
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Abstract

In this unit, protocols to culture human platelet progenitors (Colony Forming Unit–Megakaryocyte; CFU‐M) with or without toxicants are described. Platelet progenitors are obtained from human umbilical cord blood. After separation of mononuclear cells, the cell suspension can be cryopreserved or plated immediately. Megakaryocytes are identified by immunocytochemistry. Test chemicals are added to the culture medium before cells are plated. Megakaryocytes are scored after 12 days of culture. IC10, IC50 and IC90 can be calculated by comparison to control cultures. A predictive model is proposed to evaluate the hazard of thrombocytopenia induced by chemicals. When IC50 and IC90 are below Cmax in humans, the likelihood of thrombocytopenia is strong. Curr. Protoc. Toxicol. 37:20.5.1‐20.5.14. © 2008 by John Wiley & Sons, Inc.

Keywords: clonogenic assays; thrombocytopenia; human CFU‐MK; predictive model

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: CFU‐MK Assay in the Presence of Toxicants
  • Support Protocol 1: Collection of Human Umbilical Cord Blood
  • Support Protocol 2: Cryopreservation and Thawing of Human Mononuclear Cord Blood Cells
  • Support Protocol 3: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: CFU‐MK Assay in the Presence of Toxicants

  Materials
  • Human cord blood mononuclear cells (HuCBMCs; Support Protocols protocol 21 and protocol 32)
  • 1× Iscove's modified Dulbecco's medium (IMDM; Invitrogen, cat. no. 21980‐032)
  • MegaCult‐C Complete Kit with Cytokines (StemCell Technologies, cat. no. 04971) including:
    • Bovine collagen solution (may be purchased individually as StemCell Technologies cat. no. 04902)
    • MegaCult‐C serum‐free medium with cytokines (may be purchased individually as StemCell Technologies cat. no. 04901, 2 ml/tube)
    • Negative control antibody: anti‐TNP (mouse anti‐trinitrophenol antibody; isotype IgG 2a)
    • Secondary antibody: biotin‐conjugated goat anti–mouse IgG
    • Avidin–alkaline phosphatase conjugate
    • Alkaline phosphatase substrate tablets
    • Evans blue counterstain
    • Human serum
    • Filter cards
  • Toxicant compounds of interest
  • Methanol (e.g., Carlo Erba, cat. no. 414816)
  • Acetone (e.g., Carlo Erba, cat. no. 400971)
  • 0.05 M Tris/NaCl buffer, pH 7.6 (see recipe)
  • MegaCult‐C Staining Kit (StemCell Technologies, cat. no. 04962) or primary antibody: anti‐GPIIb/IIIa (isotype IgG 2a)
  • 10% (w/v)BSA solution
  • 15‐ml round‐bottom tubes (Falcon, cat. no. 2057)
  • Double‐chamber slides (StemCell Technologies; cat. no. 04813) or Lab‐Tek chamber slide system (Nunc; cat. no.177429)
  • Inverted microscope with 20× to 40× magnification
  • 2.5‐liter plastic container (26‐cm × 16‐cm × 6‐cm)
  • Polypropylene spacers
  • 65‐mm gridded tissue culture dish

Support Protocol 1: Collection of Human Umbilical Cord Blood

  Materials
  • Citrate‐phosphate‐dextrose (CPD) solution (Sigma, cat. no. C‐7165)
  • Human umbilical cord after normal delivery
  • 10× Dulbecco's phosphate‐buffered saline without calcium and magnesium. (CMF‐DPBS; Invitrogen, cat. no. 14200); dilute 1/10 just before use with sterile H 2O for cell culture (Eurobio, cat. no. 010162) or sterile Milli‐Q H 2O; alternatively purchase 1× CMF‐DPBS (Invitrogen, cat. no. 14190)
  • Ficoll‐Paque, research grade (GE Healthcare, cat. no. 17‐0840‐02)
  • Trypan blue solution: HBSS (Sigma; H2513) containing 0.4% (w/v) trypan blue (Sigma; T8154)
  • Turk solution: HBSS (Sigma; H2513) containing 2% (v/v) acetic acid (Sigma; A6284) and 0.01% (w/v) methylene blue (Sigma; M9140)
  • 15‐ml conical tubes (Falcon; 2097) for density gradient
  • Centrifuge
  • 50‐ml conical tubes (Falcon; cat. no. 2070)
  • Additional reagents and equipment for counting cells in a hemacytometer and counting viable cells by trypan blue exclusion ( appendix 3B)

Support Protocol 2: Cryopreservation and Thawing of Human Mononuclear Cord Blood Cells

  Materials
  • 1× Iscove's modified Dulbecco's medium (IMDM; Invitrogen, cat. no. 21980‐032 or 21980‐024)
  • Dimethylsulfoxide (DMSO; Sigma; D5679)
  • Fetal bovine serum (FBS; if possible, pretested; Invitrogen, cat no. 10084‐150)
  • Liquid N 2
  • Human mononuclear cord blood cells to be frozen ( protocol 2)
  • Human albumin for clinical applications, Fraction V, 96% to 99% albumin (Sigma, cat. no. A1653)
  • Dextran T40 (GE Healthcare, cat no. 17‐0270‐01)
  • 10 U/µl DNase I, RNase free (Boehringer, cat. no. 776785)
  • Trypan blue solution: HBSS (Sigma; H2513) containing 0.4% (w/v) trypan blue (Sigma; T8154)
  • Turk solution: HBSS (Sigma; H2513) containing 2% (v/v) acetic acid (Sigma; A6284) and 0.01% (w/v) methylene blue (Sigma; M9140)
  • Cryotubes: 1.8‐ml (Nunc: cat. no. 377267), 2.0‐ml (Greiner; cat. no. 9597), or 1.2‐ml (Falcon; cat. no. 4806)
  • Freezing container, e.g., “Mr. Frosty” (Nalgene; cat. no. 5100)
  • Liquid N 2 freezer
  • 0.22‐µm sterile filters
  • 15‐ml and 50‐ml conical tubes (Falcon; cat. no. 2070 and 2097, respectively)
  • 100‐µm cell strainer (Falcon; cat. no. 2360)
  • Centrifuge
  • Additional reagents and equipment for counting cells in a hemacytometer and counting viable cells by trypan blue exclusion ( appendix 3B)
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Figures

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Literature Cited

Literature Cited
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