Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also known as SWELL1, subunit of the VRAC channel. The second goal is to determine if an increase in the number of VRAC channels at the cell membrane is responsible for an increase in chloride permeability of the membrane in two well-known cases: during staurosporine (STS)-induced apoptosis and after water balance disturbance caused by hypotonic medium. Human suspension lymphoid cells U937 were used as they are suitable for flow fluorometry and because we have recently studied their membrane chloride permeability during apoptosis. We found that surface expression of endogenous LRRC8A subunits can be quantified in living U937 cells using flow fluorometry with the Alomone Lab antibody. Further, we revealed that treatment of cells for 1 hour using STS or a hypotonic solution did not change the number of LRRC8A subunits to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis. This indicates that prolonged increase in chloride permeability of the cell membrane during apoptotic cell shrinkage or cell volume regulation under hypotonicity in U937 cells occurs without altering cell surface expression of VRAC.

中文翻译：

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在活U937细胞膜上阴离子通道VRAC亚基LRRC8A的流式荧光定量分析。

评估细胞膜上通道的表达是研究活细胞中离子通道功能的必要步骤。首先，我们探讨是否可以使用流式细胞仪和针对LRRC8A，也称为SWELL1，VRAC通道亚基的细胞外环的市售抗体测定内源性VRAC。第二个目标是确定在两种众所周知的情况下，细胞膜上VRAC通道数量的增加是否导致膜的氯离子渗透性增加：在星形孢菌素（STS）诱导的细胞凋亡期间以及在水平衡紊乱之后由低渗介质引起。使用人类悬浮淋巴样细胞U937是因为它们适用于流式荧光分析，并且因为我们最近研究了其在凋亡过程中的膜氯化物渗透性。我们发现，可以使用Alomone Lab抗体使用流式荧光法在活U937细胞中定量内源LRRC8A亚基的表面表达。此外，我们揭示了使用STS或低渗溶液处理细胞1小时不会将LRRC8A亚基的数量改变到与通过离子含量分析确定的膜氯化物渗透性变化相对应的程度。这表明在U937细胞中低渗下凋亡细胞收缩或细胞体积调节期间，细胞膜的氯化物通透性延长了，而没有改变VRAC的细胞表面表达。我们发现使用STS或低渗溶液处理细胞1小时不会改变LRRC8A亚基的数量，其变化程度与通过离子含量分析确定的膜氯化物渗透性的变化相对应。这表明在U937细胞中低渗下凋亡细胞收缩或细胞体积调节期间，细胞膜的氯化物通透性延长了，而没有改变VRAC的细胞表面表达。我们发现，使用STS或低渗溶液处理细胞1小时不会改变LRRC8A亚基的数量，其变化程度与通过离子含量分析确定的膜氯化物渗透性的变化相对应。这表明在U937细胞中低渗下凋亡细胞收缩或细胞体积调节期间，细胞膜的氯化物通透性延长了，而没有改变VRAC的细胞表面表达。