Fluorescence-based measurement of nitric oxide synthase activity in activated rat macrophages using dichlorofluorescin
Fluorescence-based measurement of nitric oxide synthase activity in activated rat macrophages using dichlorofluorescin
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DOI：
10.1006/niox.1997.0135
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发表时间：
1997-08-01
影响因子：
3.9
通讯作者：
Kobzik, L
中科院分区：
文献类型：
--
作者：
Imrich, A;Kobzik, L
我们研究了氧化反应性探针二氯荧光素（DCFH）用于测定干扰素-γ（IFN-γ）激活的大鼠肺泡巨噬细胞（AM）中NO合酶（NOS）活性的实用性（IFN-γ，25 U/ml），使用基于微孔板的荧光测定法和流式细胞术分析，接受l治疗的AM（而不是d）-精氨酸显示DCFH氧化的剂量和时间依赖性增加高于缓冲液对照（例如，DCF产生（nM）：对照，d-精氨酸100 μ M，l-精氨酸100 μ M; 91 +/- 9，76 +/- 11，396 +/- 45，平均值+/- SE，n = 6，120 min）。此外，NOS抑制剂（硝基-L-精氨酸）显示完全抑制L-精氨酸依赖性DCF的产生。平行测定显示在相同样品中DCFH氧化与亚硝酸盐产生的强相关性（例如，DCF产量（nM）：143，222，409;亚硝酸盐（μ M）：2.2，3.6，7.1;与此相反，交替的氧化剂反应性探针氢乙啶（HE）和二氢罗丹明（DHR）没有报道与L-精氨酸孵育的活化AM中氧化剂产生增加，尽管它们能够容易地检测用甲萘醌（100 μ M）处理的细胞中细胞内超氧阴离子的产生。我们的结论是，DCFH是一个有用的探针，用于定量激活大鼠肺巨噬细胞中的NOS-2活性。(C)北京：科学出版社.
We investigated the utility of the oxidant-reactive probe dichlorofluorescin (DCFH) for measurement of NO synthase (NOS) activity in rat alveolar macrophages (AMs) activated by culture for 18 h with interferon-gamma (IFN-gamma, 25 U/ml), Using both microplate-based fluorometry and flow cytometric analysis, AMs treated with l (but not d)-arginine showed a dose-and time dependent increase in DCFH oxidation above buffer control (e,g,, DCF production (nM): control, d-arginine 100 uM, l-arginine 100 mu M respectively; 91 +/- 9, 76 +/- 11, 396 +/- 45, mean +/- SE, n = 6, 120 min), Furthermore, the NOS inhibitor (nitro-l-arginine) showed complete inhibition of the l-arginine-dependent DCF production. Parallel assays showed a strong correlation in DCFH oxidation with nitrite production in the same samples (e.g., DCF production (nM): 143, 222, 409; nitrite (mu M): 2.2, 3.6, 7.1; for 5, 10, 50 mu M I-arginine, respectively), In contrast, the alternate oxidant-reactive probes hydroethidine (HE) and dihydrorhodamine (DHR) did not report increased oxidant production in activated AMs incubated with l-arginine, despite their ability to easily detect intracellular superoxide anion production in cells treated with menadione (100 mu M). We conclude that DCFH is a useful probe for quantitation of NOS-2 activity in activated rat lung macrophages. (C) 1997 Academic Press.