Flow Cytometry of the Side Population (SP)

Jordi Petriz1

1 Vall d'Hebron Institut de Recerca, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.23
DOI:  10.1002/0471142956.cy0923s64
Online Posting Date:  April, 2013
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Abstract

The side population (SP) has become an important hallmark for the definition of the stem‐cell compartment, especially for the detection of stem cells and for their physical isolation by fluorescence‐activated cell sorting (FACS). SP cells are CD34 and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, the protocol is difficult for most investigators to perform: first, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because analysis of the acquired data requires extensive expertise in flow cytometry to accurately detect the SP events. More recently, a new assay based on the efflux of Vybrant DyeCycle Violet stain (DCV) has been documented to discriminate SP cells. This unit contains many helpful pointers to aid the user in obtaining the best possible results with these assays. Curr. Protoc. Cytom. 64:9.23.1‐9.23.20. © 2013 by John Wiley & Sons, Inc.

Keywords: side population; stem cells; CD 34 negative; Hoechst 33342; Vybrant DyeCycle Violet stain; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Gating Strategy and Sample Acquisition for Flow Cytometric Identification of Side‐Population Stem Cells
  • Support Protocol 1: Sample Preparation for Flow Cytometry of the Side Population
  • Support Protocol 2: Labeling of Viable Cells for Flow Cytometry of the Side Population
  • Support Protocol 3: Simultaneous Staining with Antibodies
  • Support Protocol 4: Laser Alignment and Quality Control
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Gating Strategy and Sample Acquisition for Flow Cytometric Identification of Side‐Population Stem Cells

  Materials
  • Stained cells (see Support Protocols protocol 32 and protocol 43)
  • For UV excitation:
    • Flow cytometer equipped with UV laser operating at ∼350 nm and standard 488‐nm laser or laser diode
    • Filter combination consisting of 405/30 band‐pass (blue), 670/40 band‐pass (red), and 440 dichroic long‐pass filters or 450/65 band‐pass (blue), 670/40 band‐pass (red) and 510 dichroic long‐pass filters
  • For Violet excitation:
    • Flow cytometer equipped with violet laser operating at ∼405 nm and standard 488‐nm laser or laser diode
    • Filter combination consisting of 450/40 band‐pass (blue), 603/48 band‐pass (red), and 500 dichroic long‐pass filters
NOTE: Keep the sample at 4°C and protected from light during analysis.

Support Protocol 1: Sample Preparation for Flow Cytometry of the Side Population

  Materials
  • Hematopoietic sample, tissue/tumor sample, or adherent cultured cells to be stained
  • Ammonium chloride lysing solution (see recipe)
  • BME‐HEPES: Basal Medium Eagle (e.g., Life Technologies) supplemented with 10 mM HEPES
  • Enzyme medium (see recipe)
  • DMEM+ (see recipe)
  • DMEM+ (see recipe) supplemented with 500 Kunitz units/ml DNase (prepare fresh daily; store at 4°C)
  • 1× Trypsin‐EDTA (0.5 mg/ml trypsin and 0.22 mg/ml EDTA in PBS without calcium and magnesium)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Refrigerated centrifuge
  • Curved‐edge scalpels
  • 75‐ml trypsinization flasks (e.g., Fisher)
  • Magnetic stirrer
  • 50‐ml conical polypropylene centrifuge tubes
  • 50‐µm nylon mesh
  • 37°C shaking incubator
  • Additional reagents and equipment for preparation of white blood cell samples for flow cytometry (unit 5.1), counting cells ( appendix 3A), and trypsinization of cultured adherent cells ( appendix 3B)

Support Protocol 2: Labeling of Viable Cells for Flow Cytometry of the Side Population

  Materials
  • DMEM+ (see recipe)
  • Sample of nucleated cells, ∼1–2 ×106 cells/ml ( protocol 2)
  • 1× ammonium chloride lysing solution (optional; see recipe for 10×)
  • Türk solution (see recipe; optional)
  • 1 mg/ml stock solution of Hoechst 33342 (Ho342; Sigma) in H 2O (filter sterilize and store in 1‐ml single‐use aliquots at −20°C)
  • 5 mM stock solution of Vybrant DyeCycle Violet stain (DCV; Life Technologies) in H 2O (store up to 6 months at 2° to 6°C, protected from light; do not freeze)
  • 5 mM stock solution of verapamil (Sigma) in H 2O (filter sterilize and store up to 1 month at 4°C) or 5 mM stock solution of fumitremorgin C (FTC; available through NIH drug screen; http://dtp.nci.nih.gov/branches/dscb/repo_request.html{\#}spec‐request) in DMSO (filter sterilize and store up to 1 month at 4°C)
  • HBSS+ (see recipe)
  • 1 mg/ml stock solution of propidium iodide (PI; Sigma) in H 2O (store up to 1 month at 4°C)
  • Light‐protected circulating water bath kept exactly at 37°C
  • Refrigerated centrifuge
  • 50‐ml conical polypropylene centrifuge tubes
  • 50‐µm nylon mesh
  • Additional reagents and equipment for simultaneous staining with antibodies (optional; protocol 4)
NOTE: All incubations should be carried out with periodic mixing. Incubations must be done in the dark, but the incubation tubes should not be covered with aluminum foil, which can inhibit heat transfer to the cells.

Support Protocol 3: Simultaneous Staining with Antibodies

  • Conjugated antibodies (BD Biosciences, Beckman Coulter, Life Technologies, Miltenyi Biotec), e.g.:
    • FITC‐CD45
    • FITC‐CD244
    • APC‐CD38
    • APC‐CD150
    • PE‐CD34
    • PE‐CD48
    • PE‐CD90
    • PE‐CD117
    • PE‐CD133
  • HBSS+ (see recipe) containing 5 µg/ml PI

Support Protocol 4: Laser Alignment and Quality Control

  Materials
  • Fluorescent beads (also see unit 1.3):
    • For Violet laser alignment: SPHERO Ultra Rainbow Calibration Particles, which are especially useful in the Red and Violet channels (Spherotec)
    • For 488‐nm laser alignment: Flow‐Check fluorospheres (Beckman Coulter)
    • For UV laser alignment: 4.5‐µm diameter, emission maximum, ∼405 nm (Fluoresbrite BB Carboxylate microspheres; Polysciences)
  • Flow cytometer with 450/40 band‐pass filter and 500 dichroic long‐pass (DLP) filter for analyzing beads
  • Flow cytometer with 405/30 band‐pass filter and 440 dichroic long‐pass (DLP) filter for analyzing beads
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Figures

  •   FigureFigure 9.23.1 Hoechst 33342 SP analysis of unpurified human bone marrow on a MoFlo cell sorter (Beckman Coulter) equipped with an Enterprise II water‐cooled ion laser emitting at 351 nm (30 mW of laser power). (A) Representative dot plot for Hoechst Blue versus Hoechst Red simultaneous emission from a human bone‐marrow sample containing SP cells. (B) Schematic dot plot showing distribution of events.
  •   FigureFigure 9.23.2 Dye Cycle Violet SP analysis of ABCG2 transfectants and human astrocytoma cells on an Attune Acoustic Focusing cytometer (Life Technologies) equipped with a 50‐mW violet laser emitting at 405 nm. Vybrant DyeCycle Violet stain (DCV), with emission characteristics similar to those of Hoechst 33342, but with a longer wavelength excitation maxima (369 nm), has been validated to study SP cells. Using DCV and violet excitacion, the SP displays almost identical distributions as for the Ho342 SP assay. (A) MXRA cells with enforced expression of ABCG2. (B) Human brain astrocytoma grade III SW 1783 cells. Cell subpopulations are as indicated in Figure B.
  •   FigureFigure 9.23.3 Hoechst 33342 SP analysis of unpurified human bone marrow on a MoFlo cell sorter (Beckman Coulter) equipped with an Enterprise II water‐cooled ion laser emitting at 351 nm. Since the red signal is lower in intensity by default (because of the intrinsic physical properties of this dye), resolution in the red emission is not as good as in the blue, and relatively higher laser power is needed. (A) Effect of UV laser misalignment on resolution of the SP. (B) Analysis of a bone‐marrow sample stained with a suboptimal source (different batch).
  •   FigureFigure 9.23.4 Hoechst 33342 SP analysis of unpurified human bone marrow. The analysis was carried out using (A) a filter setup consisting of BP 405/30 (blue), BP 670/40 (red), and 440 DLP filters, and (B) an alternative combination consisting of BP 450/65 (blue), BP 670/40 (red), and 510 DLP filters.
  •   FigureFigure 9.23.5 Side population (SP) pattern of Hoechst 33342 efflux in the bone marrow of a patient with acute myeloid leukemia (AML) at diagnosis. Using Hoechst 33342 dye and dual‐wavelength flow cytometry analysis, an SP profile is found to be present in AML samples. The SP contains increasing levels of ABCG2‐mediated dye efflux, from high to low fluorescence. (A) A representative flow cytometry dot plot of diploid and near‐tetraploid AML (M2) cells also stained with Hoechst 33342 to identify SP+ cells (diploid SP+ cells: 13.31%; near‐tetraploid SP+ cells: 1.89%). (B) DNA content through high‐resolution cell cycle analysis, corresponding to the patient diagnosed with AML (M2), showing a secondary near‐tetraploid subpopulation (DNA index = 2.1) with individual S+G2M phase, as calculated from the linear relationship between DNA content and propidium iodide fluorescence.
  •   FigureFigure 9.23.6 Side population (SP) pattern of Hoechst 33342 efflux in ABCB1 expressing cells on a MoFlo cell sorter (Beckman Coulter) equipped with an Enterprise II water‐cooled ion laser emitting at 351 nm (30 mW of laser power). Hoechst 33342 SP analysis of (A) CD34+ KG1a human leukemic cells and (B) human purified CD34+ normal cells, with constitutive expression of ABCB1. KB3.1 cells stably transfected with the V185 mutant ABCB1, incubated with Ho342 in absence (C) and presence (D) of the ABCB1 inhibitor, verapamil.

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Literature Cited

Literature Cited
   Abbott, B.L., Colapietro, A.M., Barnes, Y., Marini, F., Andreeff, M., and Sorrentino, B.P. 2002. Low levels of ABCG2 expression in adult AML blast samples. Blood 100:4594‐4601.
   Balbuena, J., Pachon, G., Lopez‐Torrents, G., Aran, J.M., Castresana, J.S., and Petriz, J. 2011. ABCG2 is required to control the Sonic Hedgehog pathway in side population cells with stem‐like properties. Cytometry A 79:672‐683.
   Benderra, Z., Faussat, A.M., Sayada, L., Perrot, J.Y., Tang, R., Chaoui, D., Morjani, H., Marzac, C., Marie, J.P., and Legrand, O. 2005. MRP3, BCRP, and P‐glycoprotein activities are prognostic factors in adult acute myeloid leukemia. Clin. Cancer Res. 11:7764‐72.
   Boesch, M., Reimer, D., Rumpold, H., Zeimet, A.G., Sopper, S., and Wolf, D. 2012. DyeCycle Violet used for side population detection is a substrate of P‐Glycoprotein. Cytometry A 81:517‐522.
   Cardarelli, C.O., Aksentijevich, I, Pastan, I., Gottesman, M.M. 1995. Differential effects of P‐glycoprotein inhibitors on NIH3T3 cells transfected with wild‐type (G185) or mutant (V185) multidrug transporters. Cancer Res. 55:1086‐1091.
   Chaudhary, P.M. and Roninson, I.B. 1991. Expression and activity of P‐glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 66:85‐94.
   Chen, A.Y., Yu, C., Gatto, B., and Liu, L.F. 1993. DNA minor groove‐binding ligands: A different class of mammalian DNA topoisomerase I inhibitors. Proc. Natl. Acad. Sci. U.S.A. 90:8131‐8135.
   Feuring‐Buske, M. and Hogge, D.E. 2001. Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34(+)CD38(‐) progenitor cells from patients with acute myeloid leukemia. Blood 97:3882‐3889.
   Goodell, M.A., Brose, K., Paradis, G., Conner, A.S., and Mulligan, R.C. 1996. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183:1797‐1806.
   Goodell, M.A., Rosenzweig, M., Kim, H., Marks, D.F., DeMaria, M., Paradis, G., Grupp, S.A., Sieff, C.A., Mulligan, R.C., and Johnson, R.P. 1997. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat. Med. 3:1337‐45.
   Jackson, K.A., Majka, S.M., Wang, H., Pocius, J., Hartley, C.J., Majesky, M.W., Entman, M.L., Michael, L.H., Hirschi, K.K., and Goodell, M.A. 2001. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J. Clin. Invest. 107:1395‐1402.
   Kerb, R. Implications of genetic polymorphisms in drug transporters for pharmacotherapy. 2006. Cancer Lett. 234:4‐33.
   Lechner, A., Leech, C.A., Abraham, E.J., Nolan, A.L., and Habener, J.F. 2002. Nestin‐positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP‐binding cassette transporter. Biochem. Biophys. Res. Commun. 293:670‐674.
   Litman, T., Brangi, M., Hudson, E., Fetsch, P., Abati, A., Ross, D.D., Miyake, K., Resau, J.H., and Bates, S.E. 2000. The multidrug‐resistant phenotype associated with overexpression of the new ABC half‐transporter, MXR (ABCG2). J. Cell Sci. 113:2011‐2021.
   Montanaro, F., Liadaki, K., Volinski, J., Flint, A., and Kunkel, L.M. 2003. Skeletal muscle engraftment potential of adult mouse skin side population cells. Proc. Natl. Acad. Sci. U.S.A. 100:9336‐9341.
   Murayama, A., Matsuzaki, Y., Kawaguchi, A., Shimazaki, T., and Okano, H. 2002. Flow cytometric analysis of neural stem cells in the developing and adult mouse brain. J. Neurosci. Res. 69:837‐847.
   Patrawala, L., Calhoun, T., Schneider‐Broussard, R., Zhou, J., Claypool, K., and Tang, D.G. 2005. Side population is enriched in tumorigenic, stem‐like cancer cells, whereas ABCG2+ and ABCG2‐ cancer cells are similarly tumorigenic. Cancer Res. 65:6207‐6219.
   Petersen, T.W., Ibrahim, S.F., Diercks, A.H., and van den Engh, G., 2004. Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342. Cytometry A 60:173‐181.
   Raaijmakers, M.H., de Grouw, E.P., Heuver, L.H., van der Reijden, B.A., Jansen, J.H., Scheper, R.J., Scheffer, G.L., de Witte, T.J., and Raymakers, R.A. 2005. Breast cancer resistance protein in drug resistance of primitive CD34+38‐ cells in acute myeloid leukemia. Clin. Cancer Res. 11:2436‐2444.
   Sales‐Pardo, I., Avendaño, A., Martinez‐Muñoz, V., Garcia‐Escarp, M., Whittle, P., Barquinero, J., Domingo, J.C., Marin, P., and Petriz, J. 2006. Flow cytometry of the side population: Tips and tricks. Cell Oncol. 28:37‐53.
   Shapiro, A.B. and Ling, V. 1997a. Extraction of Hoechst 33342 from the cytoplasmic leaflet of the plasma membrane by P‐glycoprotein. Eur. J. Biochem. 250:122‐129.
   Shapiro, A.B., and Ling, V. 1997b. Effect of quercetin on Hoechst 33342 transport by purified and reconstituted P‐glycoprotein. Biochem. Pharmacol. 53:587‐596.
   Shapiro, A.B., and Ling, V. 1997c. Positively cooperative sites for drug transport by P‐glycoprotein with distinct drug specificities. Eur. J. Biochem. 250:130‐137.
   Shapiro, A.B., Corder, A.B., and Ling, V. 1997. P‐glycoprotein‐mediated Hoechst 33342 transport out of the lipid bilayer. Eur. J. Biochem. 250:115‐121.
   Storms, R.W., Goodell, M.A., Fisher, A., Mulligan, R.C., and Smith, C. 2000. Hoechst dye efflux reveals a novel CD7(+)CD34(‐) lymphoid progenitor in human umbilical cord blood. Blood 96:2125‐2133.
   Teldford, W.G., Bradford, J., Godfrey, W., Robey, R.W., and Bates, S.E. 2007. Side population analysis using a violet‐excited cell‐permeable DNA binding dye. Stem Cells 25:1029‐1036.
   Uchida, N., Fujisaki, T., Eaves, A.C., and Eaves, C.J. 2001. Transplantable hematopoietic stem cells in human fetal liver have a CD34(+) side population (SP) phenotype. J. Clin. Invest. 108:1071‐1077.
   Wulf, G.G., Wang, R.Y., Kuehnle, I., Weidner, D., Marini, F., Brenner, M.K., Andreeff, M., and Goodell, M.A. 2001. A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood 98:1166‐1173.
   Zhou, S., Schuetz, J.D., Bunting, K.D., Colapietro, A.M., Sampath, J., Morris, J.J., Lagutina, I., Grosveld, G.C., Osawa, M., Nakauchi, H., and Sorrentino, B.P. 2001. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side‐population phenotype. Nat. Med. 7:1028‐1034.
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