Assessing Cell Fusion and Cytokinesis Failure as Mechanisms of Clone 9 Hepatocyte Multinucleation In Vitro

Damir Simic1, Catherine Euler1, Christina Thurby1, Mike Peden1, Sarah Tannehill‐Gregg1, Todd Bunch1, Thomas Sanderson1, Terry Van Vleet1

1 Drug Safety Evaluation, Bristol‐Myers Squibb Co., Mount Vernon, Indiana
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.9
DOI:  10.1002/0471140856.tx1409s53
Online Posting Date:  August, 2012
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Abstract

In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed‐color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell‐impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed‐color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual‐labeled multinucleated cells have resulted from fusion. Curr. Protoc. Toxicol. 53:14.9.1‐14.9.17. © 2012 by John Wiley & Sons, Inc.

Keywords: multinucleation; hepatocyte; Clone 9; cytokinesis; cell fusion

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Hepatocyte Treatment Conditions and Culturing
  • Support Protocol 1: siRNA Knockdown of Genes to Refine Mechanism
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Hepatocyte Treatment Conditions and Culturing

  Materials
  • Clone 9 cell medium: F‐12K medium (ATCC, cat. no. 30‐2004) containing 10% fetal bovine serum (FBS, e.g., Invitrogen, cat no. 16000‐044) and 1× penicillin/streptomycin (Invitrogen, cat. no. 15070‐063)
  • 75% (v/v) ethanol
  • Hepatocytes: Clone 9 cells (ATCC, cat. no. CRL‐1439)
  • 0.25% trypsin‐EDTA (Invitrogen, cat no. 25200‐056)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Vybrant CFDA SE Cell Tracer Kit—green cell marking (Invitrogen, cat. no.V12883)
  • Red Cell Tracker CMPTX—red cell marking (Invitrogen, cat. no. C34552)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Dimethylsulfoxide (DMSO; (e.g., Calbiochem, cat no. 317275)
  • 0.4% trypan blue (Sigma, cat no. T8154)
  • Drug treatments:
    • Rifabutin (Sigma, cat no. R3530)
    • Erythromycin stearate (Sigma, cat no. E9256)
    • Aurora kinase inhibitor (OM137, Sigma, cat no. O8140)
    • Rifampicin (Sigma, cat no. R3501)
    • Tamoxifen (Sigma, cat no. T5648)
    • Puromycin (Sigma, cat no. P7255)
    • siRNA (see protocol 2)
  • 4% (w/v) paraformaldehyde: dilute from 16% paraformaldehyde aqueous solution (Electron Microscopy Sciences, cat. no. 15710) as described under step 32, below
  • Antifade reagent with DAPI (Invitrogen, cat. no. S36938)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI)
  • Clear nail polish
  • 25‐ and 75‐cm2 cell culture flasks with vented caps (e.g., BD Falcon)
  • Sterile, filtered pipet tips (2‐ to 1000‐µl)
  • 15‐ml conical tubes (e.g., BD Falcon)
  • Centrifuge
  • Upright cell culture microscope or alternative device for cell counting
  • Hemacytometer (e.g., Incyto C‐Chip disposable, http://www.incyto.com/)
  • 6‐well dish
  • Microscope slides
  • Coverslips, sterile, to fit in wells of 6‐well dish
  • Confocal or fluorescent microscope
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a 37°C, 5% CO 2 humidified incubator unless otherwise indicated.

Support Protocol 1: siRNA Knockdown of Genes to Refine Mechanism

  Materials
  • AurkB siRNA (Ambion, cat. no. AM16708; siRNA ID #201286; rat target gene symbol: Aurkb)
  • Control siRNA—non silencing (ABI/Ambion, cat no. 4390843)
  • Nuclease‐free H 2O (available from most molecular biology suppliers)
  • F‐12K medium (ATCC, cat. no. 30‐2004), serum free
  • Clone 9 cells (ATCC, cat. no. CRL‐1439) growing in culture (see protocol 1)
  • HiPerFect transfection reagent (Qiagen, cat. no. 301704)
  • Clone 9 cell medium: F‐12K medium (ATCC, cat. no. 30‐2004) containing 10% fetal bovine serum (FBS, e.g., Invitrogen, cat no. 16000‐044)
  • AurkB primer/probe set (ABI, TaqMan ID Rn01460656_m1)
  • Eukaryotic 18S rRNA endogenous control Primer/Probe set (ABI, cat no. 4333760F)
  • QScript cDNA Super Mix (Quanta, cat. no. 101414)
  • PerFecta qPCR Fast Mix ROX (Quanta cat. no. 101419)
  • RNA isolation kit: e.g., RNeasy kit (Qiagen) or Cells‐to‐Ct kit (Ambion)
  • RNaseZap (Ambion, cat no. AM9780; AM9782)
  • NanoDrop Spectrophotometer ND‐1000
  • BioRad iCycler or Applied Biosystems 7900 PCR system
  • Additional reagents and equipment for quantitation of RNA using NanoDrop spectrophotometer (Desjardins and Conklin, ) and RT‐PCR (Arany, )
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Figures

Videos

Literature Cited

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