Granule enzyme exocytosis assay for cytotoxic T lymphocyte activation.
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, beta-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 beta-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.
Taffs
Rolf
R
Food and Drug Administration, Bethesda, Maryland.
Sitkovsky
Michail
M
National Institute of Allergy and Infectious Diseases, Bethesda, Maryland.
Takayama
Hajime
H
Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan.
D000818:Animals; D003124:Colorimetry / Q000379:methods*; D003594:Cytoplasmic Granules / Q000378:metabolism; D005470:Fluorometry; D005965:Glucuronates; D005966:Glucuronidase / Q000032:analysis / Q000378:metabolism; D020719:Glucuronides; D007118:Immunoassay; D008213:Lymphocyte Activation; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
D005965:Glucuronates; D020719:Glucuronides; D005966:Glucuronidase