Cell Activation Analysis by Measurement of Intracellular ph of Each Cell Subset using Flow Cytometry

众所周知，细胞内pH值（pHi）与细胞活化密切相关，如果细胞被激活，pHi通常会升高。本研究的目的是建立一套能够利用流式细胞术评估每个细胞亚群pHi的系统，并将其有效地用于细胞的生理或病理功能分析。(1)健康人外周血基础分析：采用密度单核细胞法和全血淋巴细胞法测定外周血淋巴细胞的微细胞pH值，发现流式细胞术可以测定pHi值。将BCECF（2', 7'-双（羧基乙基）5,6-羧基荧光素）作为pH指示剂加载到细胞上，并用尼日利亚菌素/钾法对细胞进行校准。(2) pHi和细胞表面抗原的双重染色用流式细胞术测量pHi最有用的优点是检查每个细胞亚群的pHi。为此，必须对pHi和细胞表面抗原进行双重染色，结果表明，负载BCECF后对细胞表面抗原进行染色效果较好，而作为荧光染料的peridinin叶绿素蛋白（peridinin叶绿素protein， perCP）效果最好。(3) pHi的时程分析采用计算机软件检测大鼠脾细胞经丝裂原（PHA和Con A）刺激后pHi的时程变化，并提出PHA和Con A活化机制的差异。(4)采用计算机软件和pHi与细胞表面抗原双染色法对各细胞亚群pHi进行时程分析。用Con A刺激大鼠脾细胞分别对CD4和CD8亚群的pHi进行时间过程分析，我们可以用这种方法测量每个细胞立方体的pHi，但Con A刺激后细胞大小变化的因素可能会影响结果，这是今后需要解决的问题。

It is well known that intracellular pH (pHi) is very closely related to cell activation and pHi is generally elevated if cells are activated. The purpose of this study is to establish the system that can evaluate pHi of each cell subset using flow cytometry and to use it effectively for analysis of physiological or pathological functions of cells.(1) Basic analysis with peripheral blood from healthy personsIntracellular pH of peripheral blood lymphocytes obtained by the method of density monocytes and lymphocytes from whole blood were both examined, and it was found that pHi measurement is possible using flow cytometry. BCECF (2', 7'-bis (carboxyethyl) 5,6-carboxy-fluorescein) was loaded on the cells as a pH indicator and cells were calibrated with the nigericin/potassium method.(2)Double staining of pHi and cell surface antigensThe most useful advatage of pHi measurement using flow cytometry is to examine pHi of each cell subset. For this purpose double stining of pHi and cell surface antigens is essential and it was found that staining of cell surface antigens after loading of BCECF was preferable and peridinin chlorophyll protein (perCP) was best as the fluorescent dye.(3) time course analysis of pHithe change in time course of pHi was examined with rat spleen cells stimulated by mitogens (PHA and Con A) using computer software, and it was suggested the difference of activation mechanism between PHA and Con A.(4) Time course analysis of pHi in each cell subsetUsing the computer software and the double staining method of pHi and cell surface antigens, respective time course analysis of pHi of CD4 and CD8 subsets was performed with rat spleen cells stimulated by Con A.We could measure the pHi of each cell cubset with this method, but the factor of change in the cell size after Con A stimulation probably affects that result and this point is the problem which should be resolved in future.