Amine‐Reactive Dyes for Dead Cell Discrimination in Fixed Samples

Stephen P. Perfetto1, Pratip K. Chattopadhyay1, Laurie Lamoreaux1, Richard Nguyen1, David Ambrozak1, Richard A. Koup1, Mario Roederer1

1 Vaccine Research Center, NIAID, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.34
DOI:  10.1002/0471142956.cy0934s53
Online Posting Date:  July, 2010
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Abstract

Amine‐reactive dyes, also known as LIVE/DEAD fixable dead cell stains, are a class of viability dyes suitable for identifying dead cells in samples that will be fixed. These dyes cross the cell membranes of dead cells, and react with free amines in the cytoplasm. Live cells exclude these dyes because their cell membranes are intact, and free dye is washed away after staining. Notably, the reaction is irreversible; therefore, when cells are fixed and permeabilized (as with intracellular staining procedures), the bound dye remains associated with the dead cells (unlike other viability dyes). Since amine‐reactive dyes are fluorescent when excited by lasers, dead cells can be identified by flow cytometry. This unit describes procedures, troubleshooting, and outcomes for using the two most commonly used amine‐reactive dyes, ViViD and Aqua Blue. Curr. Protoc. Cytom. 53:9.34.1‐9.34.14. © 2010 by John Wiley & Sons, Inc.

Keywords: amine reactive dye; LIVE/DEAD fixable cell stain; cell viability; nonspecific binding

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Titration of Amine‐Reactive Dyes
  • Basic Protocol 2: Creating Stable Amine Reactive Dye‐Labeled Compensation Beads
  • Basic Protocol 3: Staining, Gating, and Analysis of Amine‐Reactive Dyes
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Titration of Amine‐Reactive Dyes

  Materials
  • Amine reactive dye kit (ViViD, LIVE/DEAD fixable violet dead cell stain or Aqua Blue, LIVE/DEAD fixable aqua dead cell stain; both from Invitrogen) containing:
    • Dimethyl sulfoxide (DMSO)
    • Lyophilized dye
  • Phosphate‐buffered saline (PBS; Becton Dickenson)
  • Cells of interest
  • Standard staining medium (see Table 9.34.1)
  • Fluorochrome‐conjugated mAb mix (e.g., anti‐CD3 Cy7APC)
    Table 9.4.1   MaterialsStandard Staining Medium Preparation

    Reagent Volume Stock concentration Final concentration
    Neonatal calf sera (HI NCS; Invitrogen) a 20 ml 100% 4%
    Sodium azide (NaN 3) 0.5ml 20% 0.02%
    RPMI 1640 medium (Invitrogen) b 174 ml n/a n/a
    Total 500 ml

     aThaw NCS and place at 56°C for 1 hr to heat inactivate (HINCS).
     bRPMI 1640 formula #00‐0327DK.
  • 37°C water bath
  • Flow cytometer (an analyzer or sorter equipped with a violet laser and optics such as shown in Fig. ; e.g., LSR‐II, Becton Dickinson; alternatively, note in the same figure that the ViViD and Aqua Blue dyes are also well excited by a UV laser)
  • Flow cytometry data analysis software (e.g., FlowJo software, Treestar)
  • Statistical analysis software (e.g., JMP software, SAS Institute)

Basic Protocol 2: Creating Stable Amine Reactive Dye‐Labeled Compensation Beads

  Materials
  • R‐NH2 Beads (SMPLX Amine active beads; Bangs Laboratories)
  • Phosphate‐buffered saline (PBS)
  • Prediluted (reconstituted vial) amine dye at the highest concentration
  • Bead storage medium (see Table 9.34.3)
  • Flow cytometer (an analyzer or sorter equipped with a violet laser and optics such as shown in Fig. ; e.g., LSR‐II, Becton Dickinson; alternatively, note in the same figure that the ViViD and Aqua Blue dyes are also well excited by a UV laser)
    Table 9.4.3   MaterialsBead Storage Medium Preparation

    Reagent Volume Stock concentration Final concentration
    Fetal bovine serum c (HIFBS; e.g., Invitrogen) 1 ml N/A 1%
    Sodium azide (NaN 3; Sigma) 400 µl 5% 0.02%
    Phosphate‐buffered saline (PBS; Becton Dickinson) 98.6 ml N/A N/A
    Total 100 ml

     cThaw FBS and place at 56°C for 1 hr to heat inactivate (HIFBS).
  • Flow cytometry data analysis software (e.g., FlowJo software, Treestar)
  • Statistical analysis software (e.g., JMP software, SAS Institute)
NOTE: For proprietary reasons, the R‐NH2 beads can only be ordered by phone, and is not available in the Bangs Laboratories catalog.

Basic Protocol 3: Staining, Gating, and Analysis of Amine‐Reactive Dyes

  Materials
  • Amine reactive dye kit (ViViD, LIVE/DEAD fixable violet dead cell stain or Aqua Blue, LIVE/DEAD fixable aqua dead cell stain; both from Invitrogen) containing:
    • Dimethyl sulfoxide (DMSO)
    • Lyophilized dye
  • Phosphate‐buffered saline (PBS)
  • 37°C water bath
  • Standard staining medium (see Table 9.34.1)
  • Flow cytometer (an analyzer or sorter equipped with a violet laser and optics such as shown in Fig. ; e.g., LSR‐II, Becton Dickinson; alternatively, note in the same figure that the ViViD Aqua Blue dye is also well excited by a UV laser)
  • Flow cytometry data analysis software (e.g., FlowJo software, Treestar)
  • Statistical analysis software (e.g., JMP software, SAS Institute)
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Figures

Videos

Literature Cited

Literature Cited
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   Clarke, R.G. and Pinder, A.C. 1998. Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers. J. Appl. Microbiol. 84:577‐584.
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   Desrues, B., Collet, B., Rame, M.P., Bourel, D., Bourguet, P., Martin, A., Delaval, P., Toujas, L., and Dazord, L., 1989. Distribution of radiolabelled monoclonal antibody Po66 after intravenous injection into nude mice bearing human lung cancer grafts. Cancer Immunol. Immunother. 30:295‐299.
   Lamoreaux, L., Roederer, M., and Koup, R. 2006. Intracellular cytokine optimization and standard operating procedure. Nat. Protoc. 1:1507‐1516.
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   Perfetto, S., Ambrozak, D., Nguyen, R., Chattopadhyay, P., and Roederer, M. 2006a. Quality assurance for polychromatic flow cytometry. Nat. Protoc. 1:1522‐1530.
   Perfetto, S., Chattopadhyay, P., Lamoreaux, L., Nguyen, R., Ambrozak, D., Koup, R., and Roederer, M. 2006b. Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry. J. Immunol. Meth. 313:199‐208.
   Riedy, M.C., Muirhead, K.A., Jensen, C.P., and Stewart, C.C. 1991. Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations. Cytometry 12:133‐139.
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