Detection of Unseparated Human Lymphocytes by Flow Cytometry

Thomas A. Fleisher1, Gerald E. Marti2

1 Warren Grant Magnuson Clinical Center National Institutes of Health, Bethesda, Maryland, 2 Center for Biologics Evaluation and Research Food and Drug Administration, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.9
DOI:  10.1002/0471142735.im0709s08
Online Posting Date:  May, 2001
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Abstract

The protocol for flow cytometry analysis presented here has been specifically developed for studies of human peripheral blood cells. In this protocol, analysis is performed on unseparated cells in whole peripheral blood, rather than on Ficoll‐Hypaque‐purified mononuclear cells. The advantage of this approach is that it requires less time, uses smaller blood volumes, and eliminates possible differential blood loss as a result of cell separation techniques. In this regard, B cell recovery using the whole blood method is significantly greater than that obtained using Ficoll‐Hypaque‐purified mononuclear cells. However, because lymphocytes generally represent a minority of peripheral cells (especially in adults), careful gating of the test samples for lymphocytes is a more critical requirement in this procedure than in other procedures using purified cells.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Whole blood
  • Monoclonal antibodies ( appendix 4A)
  • Lysing solution
  • recipePhosphate‐buffered saline (PBS; appendix 2A) containing 0.1% sodium azide
  • 1% paraformaldehyde, pH 7.4
  • Phosphate‐buffered saline, filtered (e.g., Hematall; Fisher Scientific)
  • 12 × 75–mm round‐bottom centrifuge tube (Falcon #2052)
  • Beckman J‐6M centrifuge with JS‐4.2 rotor (or equivalent)
  • Additional reagents and equipment for drawing blood ( appendix 3A), immunofluorescence staining of cells (unit 5.3), flow cytometry (units 5.1 & 5.4), and analysis of data (unit 5.2)
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Figures

Videos

Literature Cited

Literature Cited
   Fleisher, T.A., Marti, G.E., and Hagengruber, C. 1988. Two‐color flow cytometric analysis of monocyte‐depleted human blood lymphocyte subsets. Cytometry 9:309‐315.
   Loken, M.R. and Stall, M.R. 1982. Flow cytometry as an analytical and preparative tool in immunology. J. Immunol.Methods 50:R85‐R112.
   Salzman, G.C., Crowell, J.M., Martin, J.C., Trujillo, T.T., Romero, A., Mullaney, P.F., and LaBauve, P.M. 1975. Cell classification by laser light scattering: Identification and separation of unstained leukocytes. Acta Cytol. 19:374‐377.
   Thompson, J.M., Gralow, J.R., Levy, R., and Miller, R.A. 1985. The optimal application of forward and ninety‐degree light scatter in flow cytometry for the gating of mononuclear cells. Cytometry 6:401‐406.
Key References
   Melamed, M.R., Mullaney, P.F., and Mendelsohn, M.L. 1990. Flow Cytometry and Sorting, 2nd ed. Wiley‐Liss, New York.
  Complete discussion of principles and applications of flow cytometry.
   National Committee for Clinical Laboratory Standards. 1990. Clinical Applications of Flow Cytometry: Quality Assurance and Immunophenotyping of Peripheral Blood Lymphocytes; Proposed Guidelines. NCCLS Document H42‐P. Villanova, Pa.
  Describes methods of flow cytometry.
   Shapiro, H.M., 1998. Practical Flow Cytometry, 2nd ed. Wiley‐Liss, New York.
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