Cytotoxicity of normal and activated rat monocytes analyzed by flow cytometry

✍ Scribed by Oliver Stehling; Veronika Grau; Birte Steiniger

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 150 KB
• Volume: 56A
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/cyto.a.10086

✦ Synopsis

Abstract

Background

The cytotoxic potential of activated monocytes might play an important role during severe systemic immune reactions and thus needs further elucidation. As established cytotoxicity tests are not suitable for this purpose, we developed a flow cytometry–based method.

Methods

During acute renal allograft rejection in the rat, monocytes were harvested by vascular perfusion and then purified by Percoll density gradient centrifugation and subsequent immunomagnetic negative selection. For comparison, natural killer (NK) cells were similarly isolated from spleen homogenates. Cytotoxicity was determined by flow cytometry using the fluorescein isothiocyanate–labeled NK‐sensitive lymphoma Yac‐1 as target. Necrotic cells were identified by propidium iodide, and apoptotic cells were identified by MC 540. Cytotoxicity was determined by the calculation of a cytotoxicity coefficient, ζ. The ζ coefficient describes the interrelation between the reciprocal proportion of target cells in a sample and the specific cytotoxicity, simultaneously allowing estimation of the contribution of contaminating NK cells.

Results

The method showed a substantial cytotoxicity of activated monocytes and indicated different or additional cytotoxic mechanisms compared with NK cells. Our assay permitted a detailed study of effector and target cells and took cytotoxicity of contaminating cells into account.

Conclusions

The method is nonradioactive, easy to perform, and thus helpful in investigating the role of monocytes in several diseases. Cytometry Part A 56A:81–88, 2003. © 2003 Wiley‐Liss, Inc.