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

使用流式细胞术测量每个细胞亚群的细胞内 ph 值进行细胞活化分析

基本信息

批准号：
04671440
负责人：
NAKAHARA Kazuhiko
金额：
$ 1.41万
依托单位：
Kyorin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04671440/
关键词：
Flow Cytometry Intracellular pH Cell Activation Cell Surface Antigens レーザー走査型顕微鏡

项目摘要

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.

众所周知，细胞内pH（pHi）与细胞活化非常密切相关，并且如果细胞被活化，则pHi通常升高。本研究的目的是建立流式细胞仪检测各细胞亚群pHi的系统，并将其有效地用于细胞生理或病理功能的分析。(1)对用单核细胞密度法获得的外周血淋巴细胞和全血淋巴细胞进行了细胞内pH值的测定，发现用流式细胞仪测定pHi是可行的。将BCECF（2 ′，7 ′-双（羧乙基）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/钾方法校准细胞。（2）pHi和细胞表面抗原的双重染色使用流式细胞术测量pHi最有用的优点是检查每个细胞亚群的pHi。为此目的，pHi和细胞表面抗原的双重染色是必要的，并且发现在加载BCECF之后细胞表面抗原的染色是优选的，并且多甲藻素叶绿素蛋白（perCP）作为荧光染料是最好的。(3)pHi时程分析用PHA和ConA刺激大鼠脾细胞，用计算机软件检测pHi时程的变化，提示PHA和ConA激活机制的不同。(4)各细胞亚群pHi的时程分析利用计算机软件和pHi与细胞表面抗原的双染色法，分别对ConA刺激的大鼠脾细胞CD_4和CD_8亚群的pHi进行时程分析。但Con A刺激后细胞大小的变化因素可能影响该结果，这一点是今后应解决的问题。