HIV-1 Fusion Assay
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Author:
Date:
2014-08-20
[Abstract] The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase–Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion ...
[摘要] HIV-1融合测定测量HIV-1生命周期中直至并包括病毒融合的所有步骤。 它依赖于将β-内酰胺酶-Vpr(BlaM-Vpr)蛋白嵌合体并入病毒体中,并且随后通过融合将该嵌合体转移到靶细胞中(图1)。 通过CCF2(一种β-内酰胺酶的荧光染料底物)的酶裂解来监测转移,将其装载到靶细胞中。 β-内酰胺酶在CCF2中的β-内酰胺环的切割将染料的荧光发射光谱从绿色(520nm)改变为蓝色(447nm)。 这种变化反映了病毒体融合,并且可以通过流式细胞术检测(图2)。
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Transfection of Human Naive CD4+ T Cells with PHA Activation and Neon Electroporation
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Author:
Date:
2013-08-05
[Abstract] Transfection of primary T cells can be challenging. This protocol describes a method to transfect primary human naive CD4+ T cells with an AP-1 luciferase reporter using low-level activation by phytohemagglutinin (PHA) and electroporation, as published (Palin et al., 2013). This technique is a modification of one previously described by our group (Cron et al., 2013). Anyone wishing to transfect murine T cells should consult the publication by Cron et al., 2013. This technique may be adapted for other primary T cell types by optimizing the Neon ...
[摘要] 原代T细胞的转染可能是具有挑战性的。 该方案描述了使用植物凝集素(PHA)和电穿孔的低水平激活,用AP-1荧光素酶报道子转染初级人初始CD4 + T细胞的方法,如公开的(Palin等人 。,2013)。 这种技术是先前由我们组描述的技术的修改(Cron等人,2013)。 任何希望转染鼠T细胞的人都应参考Cron等人2013年的出版物。该技术可以通过优化氖电穿孔条件来适应其它原代T细胞类型,如文中所述。 可以使用其他荧光素酶或GFP报道分子,并且将需要优化该特定报道分子的刺激条件。
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