Phenotypic Analysis Using Very Small Volumes of Blood

James L. Weaver1, Katherine McKinnon2, Dori R. Germolec3

1 Division of Applied Pharmacology Research, Office of Testing and Research, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, 2 Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 3 Toxicology Branch, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.30
DOI:  10.1002/0471142956.cy0630s54
Online Posting Date:  October, 2010
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Abstract

Analysis of cell‐surface phenotype of peripheral blood leukocytes is one of the most common applications of flow cytometry. In mouse research, the small size of the animal limits the amount of blood available. Standard staining methods using lysis of erythrocytes or gradient separation followed by repeated washing involve unavoidable losses of cells that generally limit analysis of blood to terminal methods. Time‐course studies, therefore, require sacrifice of groups of mice at each time point. Thus, a method is needed that can be used with much smaller volumes of blood. This will allow serial sampling of the same animal over time, decreasing experimental variability and reducing animal use. The method described here is a no‐lyse, no‐wash method that uses triggering on a fluorescence parameter. The method allows routine analysis of the phenotype of peripheral blood leukocytes using whole‐blood volumes of 20 µl per tube. The data are comparable with values from traditional methods requiring much higher volumes of blood. Due to interference by erythrocytes, light‐scatter parameters are not usable with this method. This method has been used for time‐course studies of peripheral blood populations in mice lasting as long as four weeks. Curr. Protoc. Cytom. 54:6.30.1‐6.30.8. © 2010 by John Wiley & Sons, Inc.

Keywords: phenotypic analysis; rodent; flow cytometry; small blood volume; leukocyte; peripheral blood leukocyte

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Phenotypic Analysis in Small Volumes of Blood
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Phenotypic Analysis in Small Volumes of Blood

  Materials
  • Antibodies directly labeled with FITC, PE or PE/Cy5 (BD Biosciences)
  • PBS‐B (see recipe)
  • Mice (as required for the experiment)
  • Anticoagulant: 0.5 M EDTA, pH 8.0 (Invitrogen; also see recipe in appendix 2A)
  • Spherotech Rainbow beads
  • Animal restrainer (e.g., Model TV‐150, Braintree Scientific)
  • Alcohol wipes
  • Syringe with 18‐G needle, sterile
  • 0.6‐ml microcentrifuge tubes
  • Veterinary adhesive (e.g., Nexaband, Abbot Laboratories)
  • 12 × 75–mm tubes
  • Flow cytometer (Coulter Elite or BD FACSort)
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Figures

Videos

Literature Cited

Literature Cited
   Bossuyt, X., Marti, G.E., and Fleisher T.A. 1997. Comparative analysis of whole blood lysis methods for flow cytometry. Cytometry 30:124‐133.
   Greve, B., Beller, C., Cassens, U., Sibrowski, W., Severin, E., and Gohde, W. 2003. High‐grade loss of leukocytes and hematopoietic progenitor cells caused by erythrocyte‐lysing procedures for flow cytometric analyses. J. Hematother. Stem Cell Res. 12:321‐330.
   Kutvirt, S.G., Lewis, S.L., and Simon, T.L. 1993. Lymphocyte phenotypes in infants are altered by separation of blood on density gradients. Br. J. Biomed. Sci. 50:213‐328.
   Pelgri, C., Rodriguez‐Palmero, M., Morante, M.P., Comas, J., Castell, M., and Franch, A. 1995. Comparison of four lymphocyte isolation methods applied to rodent T cell subpopulations and B cells. J. Immunol. Meth. 187:265‐271.
   Weaver, J.L., Broud, D.D., McKinnon, K., and Germolec, D.R. 2002. Serial phenotypic analysis of mouse peripheral blood leukocytes. Toxicol. Mech. Meth. 12:95‐118.
   Weaver, J.L., Snyder, R., Knapton, A., Herman, E.H., Honchel, R., Miller, T., Espandiari, P., Smith, R., Gu, Y.‐Z., Goodsaid, F.M., Rosenblum, I.Y., Sistare, F.D., Zhang, J., and Hanig, J. 2008. Biomarkers in peripheral blood associated with vascular injury in Sprague‐Dawley rats treated with the phosphodiesterase IV inhibitors SCH 351591 or SCH 534385. Toxicol. Pathol. 36:840‐849.
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