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

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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.