Titration of Fluorochrome‐Conjugated Antibodies for Labeling Cell Surface Markers on Live Cells

Ruud Hulspas1

1 Cytonome/ST, Boston, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.29
DOI:  10.1002/0471142956.cy0629s54
Online Posting Date:  October, 2010
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Abstract

Nonspecific antibody binding is best eliminated by optimizing the amount and concentration of the antibody. An antibody titration assay should be applied to determine the antibody amount and concentration resulting in the highest signal of the positive population and the lowest signal of the negative population. While conventional antibody titration protocols focus on the concentration of the antibody, this protocol for antibody titration considers the antibody concentration, as well as the antibody amount. Thus, it is designed to find the optimal antibody concentration for labeling antigens expressed on the surface membrane of live cells, while nonspecific antibody binding is kept to a minimum. Curr. Protoc. Cytom. 54:6.29.1‐6.29.9. © 2010 by John Wiley & Sons, Inc.

Keywords: background; nonspecific binding; monoclonal antibodies; flow cytometry; cell sorting

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Stock solution of fluorochrome‐conjugated antibody to be titered
  • Target cells for antibody titration
  • Phosphate‐buffered saline (PBS; e.g., BioWhittaker, cat. no. 17‐516Q) or equivalent
  • 1.2‐ml microcentrifuge tubes
  • 2‐, 10‐, 20‐, 100‐, 200‐, 1000‐µl pipets (e.g., Gilson Pipetman)
  • Pipet tips
  • Microcentrifuge
  • 12 × 75–mm polystyrene (or polypropylene) test tubes
  • Flow cytometer
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Figures

Videos

Literature Cited

Literature Cited
   Hulspas, R., O'Gorman, M.R.G., Wood, B.L., Gratama, J.W., and Sutherland, D.R. 2009. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B 76:355‐364.
   Kantor, A.B. and Roederer, M. 1996. FACS analysis of leukocytes. In Handbook of Experimental Immunology, 5th ed. (L.A. Herzenberg, D.M. Weir, and C. Blackwell, eds.) pp. 49.1‐49.13. Blackwell Scientific, Cambridge, Mass.
   Owens, M.A. and Loken, M.R. 1997. Titering antibodies. In Flow cytometry principles for clinical laboratory practice, pp. 65‐67. Wiley‐Liss, New York.
   Srivastava, P., Sladek, T.L., Goodman, M.N., and Jacobberger, J.W. 1992. Streptavidin based quantitative staining of intracellular antigens for flow cytometric analysis. Cytometry 13:711‐721.
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