Unit

UNIT 6.30 Phenotypic Analysis Using Very Small Volumes of Blood

  1. James L. Weaver1,
  2. Katherine McKinnon2,
  3. Dori R. Germolec3

Published Online: 1 OCT 2010

DOI: 10.1002/0471142956.cy0630s54

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Weaver, J. L., McKinnon, K. and Germolec, D. R. 2010. Phenotypic Analysis Using Very Small Volumes of Blood. Current Protocols in Cytometry. 54:6.30:6.30.1–6.30.8.

Author Information

  1. 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. 2

    Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

  3. 3

    Toxicology Branch, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

Publication History

  1. Published Online: 1 OCT 2010
  2. Published Print: OCT 2010

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