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Applications of fixed-time flow cytometry in cell biology

机译：固定时间流式细胞术在细胞生物学中的应用

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Abstract: Flow cytometric (FCM) measurement of intracellular freecalcium $LB@Ca$+2$PLU$/$RB$-i$/, transients is usuallydone by two methods: (a) after a short prerun period toassess the baseline the measurement is stopped,stimulus is added and the measurement continued or (b)stimulus is injected during measurement and the samplepressure briefly increased to deliver cells rapidly tothe detection point. In (a) measurement of very shorttransients $LB@Ca$+2$PLU$/$RB$-i$/ is impeded by thelag time between stimulus addition and restart ofacquisition. In (b) response of pressure sensitivecells is hard to analyze. Furthermore, (a) and (b) donot allow to quantify and sort rare responders. Asimple Fixed-Time device has been developed.Ca$+2$PLU$/ sensitive fluorescent dye labeled cells anda stimulus are placed in different vials. Both fluidsare forced by the same pressure through tubing thatmerges into a T-junction where they mix and aredelivered through a connecting tube to the FCM:$LB@Ca$+2$PLU$/$RB$-i$/ is measured at a certain timeafter stimulation that is adjusted by sample flow rateand length of the connecting tube. With Fixed-Time, thepressure sensitive neuronal NH15-CA2 cell was analyzed.Furthermore, rare neurotransmitter responsivefibroblast from normal and transfected cultures weresorted and cloned and their dose responsecharacterized. The results demonstrate that fixed- timeFCM is an important tool for the analysis of the cellsphysiology and the preparation of responders. !23
机译：摘要：流式细胞术（FCM）测量细胞内游离钙$ LB @ Ca $ + 2 $ PLU $ / $ RB $ -i $ /，瞬态通常通过两种方法完成：（a）经过短暂的运行前评估基线测量停止，添加刺激物并继续进行测量，或（b）在测量过程中注入刺激物，短暂增加样品压力以将细胞快速递送至检测点。在（a）中，非常短瞬态的测量$ LB @ Ca $ + 2 $ PLU $ / $ RB $ -i $ /受添加刺激与重新开始获取之间的滞后时间的阻碍。在（b）中，压敏细胞的响应难以分析。此外，（a）和（b）不允许对稀有响应者进行量化和分类。已开发了Asimple固定时间设备，将Ca + 2 $ PLU $ /敏感的荧光染料标记的细胞和刺激物放在不同的小瓶中。两种流体在相同的压力下通过合并成T形结的管道被迫，在其中混合并通过连接管输送到FCM：$ LB @ Ca $ + 2 $ PLU $ / $ RB $ -i $ /刺激后一定时间，该时间可通过样品流速和连接管的长度进行调整。使用固定时间分析压敏神经元NH15-CA2细胞。此外，对正常和转染培养物中稀有的神经递质反应性成纤维细胞进行分选和克隆，并表征其剂量反应。结果表明，固定时间的FCM是分析细胞生理学和制备反应物的重要工具。 ！23

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