| Fluorescent Detection of Intracellular Nitric Oxide in Staphylococcus aureus
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Author:
Date:
2016-07-20
[Abstract] Nitric Oxide (NO) is a highly-reactive radical gas that can modify a variety of cellular targets in both eukaryotes and bacteria. NO is produced endogenously by a wide variety of organisms: For example, as a cell-signaling molecule in mammals and bacteria via nitric oxide synthase (NOS) enzymes, and as a product of denitrification. As such, it is of great benefit to NO researchers to be able to sensitively detect intracellular NO and stable reactive nitrogen species (RNS) derived from NO. To this end, a protocol for fluorescent detection of intracellular NO/RNS in biofilm cultures of the Gram-positive pathogen Staphylococcus aureus has been optimized using the commercially-available cell-permeable fluorescent stain 4-Amino-5-Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM ...
[摘要] 一氧化氮（NO）是一种高反应性的自由基气体，其可以修饰真核生物和细菌中的多种细胞靶标。 NO由多种生物体内源性产生：例如，作为哺乳动物和细菌中的细胞信号分子，通过一氧化氮合酶（NOS）酶，以及作为脱氮的产物。因此，NO研究人员能够敏感地检测来自NO的细胞内NO和稳定的活性氮物质（RNS）是非常有益的。为此，已经使用商业上可获得的细胞可渗透的荧光染料4-氨基-5来优化用于荧光检测革兰氏阳性病原体金黄色葡萄球菌的生物膜培养物中的细胞内NO/RNS的方案 - 甲基氨基-2'，7'-二氟荧光素二乙酸酯（DAF-FM二乙酸酯）。该化合物扩散到细胞中并且通过酯酶的细胞内裂解释放弱荧光DAF-FM，其与NO或其它特异性RNS反应以变成高度荧光的（Kojima等人，1999）。虽然使用荧光板读数器进行荧光的定量，但设想该方案可适用于S的细胞内NO/RNS成像。 aureus biofilms by confocal microscopy。同样，这种技术可以被优化用于检测其它生长条件（即浮游生物培养物）和/或其它细菌/古细菌中的细胞内NO/RNS。
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| Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation – A Short Protocol
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Author:
Date:
2013-12-05
[Abstract] Investigations of the activation processes involved in human monocytes and monocyte-derived macrophages and dendritic cells often required large numbers of cells that have not been possibly altered or activated by adherence to surfaces, by binding of antibodies to surface antigens during positive selection, or by release of activators by platelets or other non myeloid cells during isolation or co-culture. Human peripheral blood monocytes as well as lymphocytes from the same blood donor can be isolated by counterflow elutriation using a modification of the technique of Lionetti et al. (1980) as described previously (Bobak et al., 1986). From a unit of blood drawn into anticoagulant, 60-120 million monocytes can be obtained. These cells are not activated and have been ...
[摘要] 涉及人单核细胞和单核细胞衍生的巨噬细胞和树突细胞的激活过程的研究通常需要大量的细胞，这些细胞不可能通过附着于表面而改变或活化，通过在阳性选择期间抗体与表面抗原的结合，或通过释放 的血小板或其他非骨髓细胞在分离或共培养期间的活化剂。 人类外周血单核细胞以及来自相同血液供体的淋巴细胞可以通过逆流淘析使用Lionetti等人（1980）的技术的修改来分离，如前所述（Bobak等人 。，1986）。 从吸入抗凝血剂的单位，可以获得60-120,000个单核细胞。 这些细胞没有被活化并且已经显示在多个研究中适当地能够差别激活。
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