Time-resolved fluorometric assay for leukocyte adhesion using a fluorescence enhancing ligand.
J Immunol Methods
; 236(1-2): 19-26, 2000 Mar 06.
Article
em En
| MEDLINE
| ID: mdl-10699576
ABSTRACT
A new 96-well microtiter plate, time-resolved fluorometric assay was developed to measure leukocyte adhesion in vitro. The assay is based on loading leukocytes with a fluorescence enhancing ligand 2,2'6', 2"-terpyridine-6,6"-dicarboxylic acid (TDA), which in its acetoxymethyl ester form readily diffuses through the cell membrane. After hydrolysis by nonspecific intracellular esterases, the impermeable TDA accumulates inside the cells. When the TDA-labeled adherent leukocytes are lysed, the ligand is released and reacts with europium present in the lysis solution to produce a highly fluorescent and stable chelate. The fluorescence signal can be measured by time-resolved fluorometry and correlates directly with the number of adherent cells. In this study, we have optimized both the TDA-labeling and adhesion assay conditions in isolated human neutrophils. Furthermore, we have compared the assay with a traditional microscopic counting method. This time-resolved fluorometric assay provides a rapid, reproducible and convenient method for the routine analysis of leukocyte adhesion.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Adesão Celular
/
Fluorometria
/
Leucócitos
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Limite:
Humans
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
2000
Tipo de documento:
Article
País de afiliação:
Finlândia