Li Wen, Chung S C Sydney
Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China.
In Vitro Cell Dev Biol Anim. 2003 Nov-Dec;39(10):413-9. doi: 10.1290/1543-706X(2003)039<0413:FCEOLF>2.0.CO;2.
The aim of this study was to establish a standard flow cytometric method to measure the phagocytic function of and intracellular hydrogen peroxide (H2O2) production by rat leukocytes. Thirty-six adult, male Sprague-Dawley rats were included in this study. Whole-blood specimens from the inferior vena cava were collected in a heparinized tube and ethylenediaminetetraacetic acid (EDTA) anticoagulated tube. The phagocytic function of and intracellular H2O2 generation by leukocytes were measured with FACS Vantage trade mark flow cytometer (Becton Dickinson, San Jose, CA), using fluorescent microspheres and dihydrorhodamine-123 as probes, respectively. Several conditions were optimized in this study, including anticoagulants (heparin and EDTA), fluorescent probes (0.75- and 1.72-microm-diameter microspheres), incubation time, and concentration of the chemicals used in the experiment. Neutrophils, monocytes, and lymphocytes could be clearly defined and separated in whole blood by flow cytometry and tested for phagocytosis and intracellular H2O2 generation without the need for further purification and handling of the cells. Intracellular H2O2 production by and phagocytic function of neutrophils and monocytes were inhibited in EDTA-anticoagulated blood compared with heparin- anticoagulated blood (P < 0.01). Neutrophils showed similar phagocytic function to 0.75- and 1.72-microm microspheres, but monocytes showed weak phagocytic activity to 1.72-microm beads compared with 0.75-microm beads (P < 0.01). In conclusion, a flow cytometric method to measure the phagocytic function of and intracellular H2O2 production by rat leukocytes has been developed. Quantitative flow cytometric analysis of rat leukocyte function is convenient and feasible and provides a reliable and rapid assay to assess phagocytosis and intracellular H2O2 production by rat neutrophils and monocytes.
本研究的目的是建立一种标准的流式细胞术方法，以测量大鼠白细胞的吞噬功能和细胞内过氧化氢（H2O2）的产生。本研究纳入了36只成年雄性Sprague-Dawley大鼠。从下腔静脉采集的全血标本分别收集于肝素化管和乙二胺四乙酸（EDTA）抗凝管中。使用荧光微球和二氢罗丹明-123作为探针，分别通过FACS Vantage商标流式细胞仪（Becton Dickinson，加利福尼亚州圣何塞）测量白细胞的吞噬功能和细胞内H2O2的生成。本研究优化了几个条件，包括抗凝剂（肝素和EDTA）、荧光探针（直径0.75和1.72微米的微球）、孵育时间以及实验中使用的化学物质的浓度。通过流式细胞术可以在全血中清晰地定义和分离中性粒细胞、单核细胞和淋巴细胞，并检测其吞噬作用和细胞内H2O2的生成，而无需对细胞进行进一步的纯化和处理。与肝素抗凝血液相比，EDTA抗凝血液中中性粒细胞和单核细胞的细胞内H2O2生成及吞噬功能受到抑制（P < 0.01）。中性粒细胞对直径0.75和1.72微米的微球表现出相似的吞噬功能，但与直径0.75微米的微球相比，单核细胞对直径1.72微米的微球表现出较弱的吞噬活性（P < 0.01）。总之，已开发出一种流式细胞术方法来测量大鼠白细胞的吞噬功能和细胞内H2O2的产生。大鼠白细胞功能的定量流式细胞术分析方便可行，为评估大鼠中性粒细胞和单核细胞的吞噬作用及细胞内H2O2的产生提供了一种可靠且快速的检测方法。
