A method for multiple sequential analyses of macrophage functions using a small single cell sample

Brazilian Journal of Medical and Biological Research

F.r.f. Nascimento¹  D. Rodríguez¹  E. Gomes¹  E.c. Fernvik¹  M. Russo¹ 

[1] ,Universidade de São Paulo Instituto de Ciências Biomédicas Departamento de ImunologiaSão Paulo SP ,Brasil

关键词: Macrophages;    Nitric oxide;    Hydrogen peroxide;    MHC II;    Method;   

DOI  :  10.1590/S0100-879X2003000900012

来源: SciELO

【 摘 要 】

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 µl 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.

【 授权许可】

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