Isolation and Applications of Prostate Side Population Cells Based on Dye Cycle Violet Efflux

Kalyan J. Gangavarapu1, Wendy J. Huss1

1 Roswell Park Cancer Institute, Buffalo, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 22.2
DOI:  10.1002/0471140856.tx2202s47
Online Posting Date:  February, 2011
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This unit describes methods for digestion of human prostate clinical specimens and dye cycle violet (DCV) staining for identification, isolation, and quantitation of radiolabeled dihydrotestosterone (DHT) retention of side population cells. The principle of the side population assay is based on differential efflux of DCV, a cell‐membrane‐permeable fluorescent dye, by cells with high ATP‐binding cassette (ABC) transporter activity. Cells with high ABC transporter activity that efflux DCV and fall in the lower left quadrant of a flow cytograph are designated as “side population” cells. This unit emphasizes tissue digestion, DCV staining, flow settings for sorting side population cells, and quantitation of radiolabeled DHT retention.Curr. Protoc. Toxicol. 47:22.2.1‐22.2.14. © 2011 by John Wiley & Sons, Inc.

Keywords: prostate; side population; dye cycle violet; ABC transporters

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Table of Contents

  • Introduction
  • Basic Protocol 1: Digestion of Human Prostate Clinical Specimens
  • Basic Protocol 2: Staining of Side Population Cells Using Dye Cycle Violet (DCV)
  • Basic Protocol 3: Fluorescence‐Activated Cell Sorting (FACS) of Side Population Cells
  • Basic Protocol 4: Quantitation of Retention of Radiolabeled Androgens (DHT)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Digestion of Human Prostate Clinical Specimens

  • Human prostate clinical specimen, freshly isolated, kept on ice in SPS‐1 solution (Organ Recovery Systems, Itasca, IL)
  • Sterile 1× phosphate‐buffered saline (PBS, Invitrogen), ice‐cold
  • Primary digestion solution (see recipe)
  • Hanks' buffered salt solution (HBSS; Invitrogen) with and without 5% fetal bovine serum (FBS; Invitrogen)
  • Secondary digestion solution (see recipe; thaw at 4°C and warm in a 37°C water bath before use)
  • 10% bleach solution (for disinfection)
  • Histopaque‐1077 (Sigma), sterile
  • Epithelial cell culture medium (Invitrogen)
  • Plastic Petri dishes (VWR)
  • Sterile scissors, forceps, tweezers, disposable scalpels, sink strainer
  • Sterilize conical flask with magnetic stir bar (50‐ 125‐ml flasks depending on amount of specimen)
  • Sterile 50‐ml centrifuge tubes (Krackeler)
  • 10‐ml wide‐tip pipet(s) (VWR)
  • BD Falcon strainer(s) with 100‐, 70‐, and 40‐µm pore size (BD Biosciences)
  • Aspiration‐filtering tubes connected to plastic vacuum flasks and secondary receptacles
  • 15‐ml centrifuge tubes (VWR)
  • 5‐, 10‐, and 25‐ml pipets (VWR)

Basic Protocol 2: Staining of Side Population Cells Using Dye Cycle Violet (DCV)

  • Digested human prostate epithelial cells (see protocol 1)
  • 5 mM Dye Cycle Violet (DCV; Invitrogen)
  • 10 mM fumitremorgin C (FTC; Alexis Biochemicals)
  • 7‐Aminoactinomycin D (7AAD; Beckman Coulter)
  • 15‐ml centrifuge tubes (VWR)
  • 5‐ml polystyrene tubes with cell strainer lids (BD Biosciences)

Basic Protocol 3: Fluorescence‐Activated Cell Sorting (FACS) of Side Population Cells

  • DCV‐stained cells for sorting (see protocol 2)
  • 5‐ml disposable borosilicate glass culture tubes (Kimble Chase)
  • BD FACS Aria II cell sorter with:
    • Violet diode laser (excitation 408 nm, Coherent Inc.), 450/40‐nm bandpass (BP) filter, and 65‐nm longpass (LP) filter for DCV
    • 488‐nm SapphireTM laser and 695/40‐nm BP filter for 7AAD
NOTE: The entire cell sorting and sample collection procedure should be performed at 4°C in a sterile environment.

Basic Protocol 4: Quantitation of Retention of Radiolabeled Androgens (DHT)

  • Side population cells (see protocol 3), non‐side population cells (see protocol 3), and unsorted epithelial cells (see protocol 2)
  • Epithelial cell culture medium (Invitrogen)
  • 10 mM fumitremorgin C (FTC; Alexis Biochemicals)
  • [3H]Dihydrotestosterone ([3H]DHT; PerkinElmer)
  • PBS (Invitrogen)
  • 2 N NaOH
  • 2 N HCl
  • Scintillation counter and fluid
  • 15‐ml centrifuge tubes (VWR)
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Literature Cited

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   Mathew, G., Timm, E.A. Jr., Sotomayor, P., Godoy, A., Montecinos, V.P., Smith, G.J., and Huss, W.J. 2009. ABCG2‐mediated DyeCycle Violet efflux defined side population in benign and malignant prostate. Cell Cycle 8:1053‐1061.
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Key References
  Mathew et al., 2009. See above.
  Initial reference for prostate tissue digestion and DCV side population analysis.
  Telford et al., 2007. See above.
  Compares Hoechst 33342 and DCV side populations.
  Zhou et al., 2001. See above.
  Demonstrates that ABCG2 is required for mouse hematopoietic side population.
   Goodell, 2005; Lin and Goodell, 2006; Telford, 2010. See above.
  Provides excellent protocols for Hoechst 33342 and DCV in side population analysis.
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