Fluorescent Cell Barcoding for Multiplex Flow Cytometry

Peter O. Krutzik1, Matthew R. Clutter1, Angelica Trejo1, Garry P. Nolan1

1 Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.31
DOI:  10.1002/0471142956.cy0631s55
Online Posting Date:  January, 2011
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Abstract

Fluorescent cell barcoding (FCB) enables high throughput, high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10‐ to 100‐fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines, as well as primary peripheral blood samples. Important technical considerations, such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis, are discussed. Curr. Protoc. Cytom. 55:6.31.1‐6.31.15. © 2011 by John Wiley & Sons, Inc.

Keywords: flow cytometry; multiplex; barcode; fluorescence; dye; high throughput

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Fluorescent Cell Barcoding with One Barcoding Dye in Cell Line
  • Support Protocol 1: Analysis and Deconvolution of FCB Data
  • Basic Protocol 2: Barcoding with Three Dyes in Primary PBMC
  • Support Protocol 2: Analysis and Deconvolution of Multiparameter FCB Data
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorescent Cell Barcoding with One Barcoding Dye in Cell Line

  Materials
  • Cell line of interest (e.g., U937)
  • Tissue culture medium (see recipe)
  • 16% (v/v) paraformaldehyde in water (PFA; EM grade, Electron Microscopy Sciences)
  • 100% methanol, cooled to 4°C
  • Barcoding dye stock solution at 5 mg/ml (amine reactive NHS/succinimidyl ester of Alexa Fluor 488, Invitrogen; see recipe)
  • Dimethyl sulfoxide (DMSO)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 14190‐144), cooled to 4°C
  • Staining medium (SM; see recipe)
  • Antibodies against intracellular antigen of interest (e.g., phospho‐protein, cytokine, transcription factor), optional
  • 37°C, 5% CO 2 incubator
  • 5‐ml polystyrene FACS tubes (BD Falcon)
  • Vortex
  • Benchtop centrifuge with swinging‐bucket rotor and 5‐ml tube carrier
  • 1.5‐ml microcentrifuge tubes
  • Flow cytometer with 488‐nm laser line (e.g., Becton Dickinson FACScan or FACSCalibur)

Support Protocol 1: Analysis and Deconvolution of FCB Data

  Materials
  • Primary peripheral blood mononuclear cells (PBMCs)
  • Tissue culture medium (see recipe)
  • 16% (v/v) paraformaldehyde (PFA; EM grade, Electron Microscopy Sciences) in water
  • 100% methanol, cooled to 4°C
  • Barcoding dye stock solution at 5 mg/ml, amine reactive NHS/succinimidyl ester of: DyLight 350 and DyLight 800 (Pierce Thermo Scientific), and Pacific Orange (Invitrogen; see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 14190‐144), cooled to 4°C
  • Staining medium (SM; see recipe)
  • Antibodies against intracellular antigen of interest (e.g., phospho‐protein, cytokine, transcription factor), optional
  • 37°C, 5% CO 2 incubator
  • 96‐well polypropylene V‐bottom deep block plate, 2‐ml capacity
  • Vortex
  • Benchtop centrifuge
  • 96‐well polypropylene U‐bottom plate
  • FACS tubes
  • Flow cytometer with 355‐, 405‐, 488‐, and 633‐nm laser line (e.g., Becton Dickinson LSRII or LSRFortessa)
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Figures

  •   FigureFigure 6.31.1 Fluorescent cell barcoding protocol. Cell samples are labeled with an amine‐reactive fluorescent dye (FCB dye) at different concentrations. After covalent labeling has occurred, cells are washed to remove unbound dye, then combined into one tube, and stained with antibodies against intracellular or surface antigens. The combined tube is then acquired on the cytometer. After acquisition, the original cell samples are identified by gating populations that display discrete fluorescent intensities in the FCB channel.
  •   FigureFigure 6.31.2 Deconvolution of four barcoded samples. Four independent tubes of U937 cells were barcoded with 0, 0.016, 0.063, or 0.25 µg/ml AlexaFluor 488 (Ax488), then combined into one tube and run on the flow cytometer. After identifying singlet events, intact cells are gated based on forward and side scatter characteristics. Plotting the FCB channel (Ax488 in this case) versus side scatter reveals four distinct populations that correspond to the four original tubes that were barcoded. Once gated, the populations can be analyzed for other antigens of interest.
  •   FigureFigure 6.31.3 Layout of barcoding matrix used to encode 27 samples. Three FCB dyes were used: DyLight 350, Pacific Orange, and DyLight 800. Each dye was used at three concentrations. DyLight 350 encoded the three rows while the combination of Pacific Orange and DyLight 800 encoded the nine columns.
  •   FigureFigure 6.31.4 Deconvolution of 27 barcoded primary cell populations. 27 individual wells were barcoded using all the unique combinations of DyLight 350 at 0, 0.5, or 2 µg/ml; Pacific Orange at 0, 0.25, or 1 µg/ml, and DyLight 800 at 0, 0.25, or 1 µg/ml. Cell events were first identified by gating on singlets (FSC‐area vs. ‐width) and cells (FSC‐area vs. SSC‐area) as in Figure . Three populations are then clearly visible when plotting any of the barcoding parameters against side scatter. In this analysis, the rows were first gated based on staining intensity in the DyLight 350 parameter. The three rows were then analyzed for Pacific Orange staining, which was used to separate the columns into groups of three. Analyzing each of the levels of Pacific Orange for DyLight 800 revealed the samples from individual wells C1‐C9. This analysis was repeated for each row, yielding 27 gated populations. The gating of the individual wells within rows A and B are not shown.

Videos

Literature Cited

Literature Cited
   Clutter, M.R., Heffner, G.C., Krutzik, P.O., Sachen, K.L., and Nolan, G.P. 2010. Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry. Cytometry A 77:1020‐1031.
   Irish, J.M., Myklebust, J.H., Alizadeh, A.A., Houot, R., Sharman, J.P., Czerwinski, D.K., Nolan, G.P., and Levy, R. 2010. B‐cell signaling networks reveal a negative prognostic human lymphoma cell subset that emerges during tumor progression. Proc. Natl. Acad. Sci. U.S.A. 107:12747‐12754.
   Krutzik, P.O. and Nolan, G.P. 2003. Intracellular phospho‐protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 55:61‐70.
   Krutzik, P.O. and Nolan, G.P. 2006. Fluorescent cell barcoding in flow cytometry allows high‐throughput drug screening and signaling profiling. Nat. Methods 3:361‐368.
   Krutzik, P.O., Crane, J.M., Clutter, M.R., and Nolan, G.P. 2008. High‐content single‐cell drug screening with phosphospecific flow cytometry. Nat. Chem. Biol. 4:132‐142.
   Schulz, K.R., Danna, E.A., Krutzik, P.O., and Nolan, G.P. 2007. Single‐cell phospho‐protein analysis by flow cytometry. Curr. Protoc. Immunol. 78:Unit 8.17.1‐8.17.20.
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