A method for multiple sequential analyses of macrophage functions using a small single cell sample.
Braz J Med Biol Res
; 36(9): 1221-6, 2003 Sep.
Article
em En
| MEDLINE
| ID: mdl-12937789
Microbial pathogens such as bacillus Calmette-Guérin (BCG) induce the activation of macrophages. Activated macrophages can be characterized by the increased production of reactive oxygen and nitrogen metabolites, generated via NADPH oxidase and inducible nitric oxide synthase, respectively, and by the increased expression of major histocompatibility complex class II molecules (MHC II). Multiple microassays have been developed to measure these parameters. Usually each assay requires 2-5 x 10(5) cells per well. In some experimental conditions the number of cells is the limiting factor for the phenotypic characterization of macrophages. Here we describe a method whereby this limitation can be circumvented. Using a single 96-well microassay and a very small number of peritoneal cells obtained from C3H/HePas mice, containing as little as <=2 x 10(5) macrophages per well, we determined sequentially the oxidative burst (H2O2), nitric oxide production and MHC II (IAk) expression of BCG-activated macrophages. More specifically, with 100 micro l of cell suspension it was possible to quantify H2O2 release and nitric oxide production after 1 and 48 h, respectively, and IAk expression after 48 h of cell culture. In addition, this microassay is easy to perform, highly reproducible and more economical.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Macrófagos Peritoneais
/
Ativação de Macrófagos
/
Óxido Nítrico
Limite:
Animals
Idioma:
En
Revista:
Braz J Med Biol Res
Ano de publicação:
2003
Tipo de documento:
Article
País de afiliação:
Brasil
País de publicação:
Brasil