Unit

UNIT 3.16 Granule Enzyme Exocytosis Assay for Cytotoxic T Lymphocyte Activation

  1. Rolf Taffs1,
  2. Michail Sitkovsky2,
  3. Hajime Takayama3

Published Online: 1 MAY 2001

DOI: 10.1002/0471142735.im0316s25

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Taffs, R., Sitkovsky, M. and Takayama, H. 2001. Granule Enzyme Exocytosis Assay for Cytotoxic T Lymphocyte Activation. Current Protocols in Immunology. 25:III:3.16:3.16.1–3.16.9.

Author Information

  1. 1

    Food and Drug Administration, Bethesda, Maryland

  2. 2

    National Institute of Allergy and Infectious Diseases, Bethesda, Maryland

  3. 3

    Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: MAR 1998

Abstract

Upon activation by specific target cells, cytotoxic T lymphocytes (CTL) release into the culture medium the content of cytoplasmic granules that contain serine esterases. The amount of enzyme released during CTL activation can be easily quantitated by spectrophotometric measurement of the colored product of the enzymatic degradation of a synthetic substrate. In the primary method presented here, CTL are activated with monoclonal antibodies prepared against the T cell receptor (TCR) complex, then activation is quantitated according to the amount of serine esterase released in the supernatant. Alternate protocols describe the activation of CTL by a combination of protein kinase C and calcium ionophores (a TCR-independent approach) and by the more conventional approach of target-cell mediation. In a third approach, β-glucuronidase rather than esterase activity is measured, as this enzyme is also present in granules released upon CTL activation. This unit therefore includes a colorimetric assay for CTL-induced β-glucuronidase activity employing the substrate phenolphthalein glucuronic acid as well as a corresponding automated fluorimetric assay employing the substrate 4-methylumbelliferyl-D-glucuronide. Finally, the quantitation of granule exocytosis resulting from cell damage or death induced by the activating agent, rather than CTL activation, is described.