UNIT 5.3 Preparation of Cells and Reagents for Flow Cytometry

  1. Kevin Holmes1,
  2. Larry M. Lantz1,
  3. B.J. Fowlkes1,
  4. Ingrid Schmid2,
  5. Janis V. Giorgi (Intracellular Staining)2

Published Online: 1 NOV 2001

DOI: 10.1002/0471142735.im0503s44

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Holmes, K., Lantz, L. M., Fowlkes, B., Schmid, I. and Giorgi, J. V. 2001. Preparation of Cells and Reagents for Flow Cytometry. Current Protocols in Immunology. 44:5.3:5.3.1–5.3.24.

Author Information

  1. 1

    National Institute of Allergy and Infectious Diseases, Bethesda, Maryland

  2. 2

    UCLA School of Medicine, Los Angeles, California

Publication History

  1. Published Online: 1 NOV 2001
  2. Published Print: AUG 2001


Flow cytometry is widely used for analyzing the expression of cell surface and intracellular molecules (on a per cell basis), characterizing and defining different cell types in heterogeneous populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. This technique is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies or ligands that bind to specific cell-associated molecules. A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. Alternate protocols describe intracellular staining of unfixed cells in the presence of a detergent and staining of nonviable cells to facilitate discrimination of dead cells in fixed or permeabilized cell preparations.