| Project/Area Number |
04671440
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory medicine
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| Research Institution | Kyorin University |
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Principal Investigator |
NAKAHARA Kazuhiko Kyorin University, Faculty of Medicine, Professor, 医学部, 教授 (70101095)
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| Co-Investigator(Kenkyū-buntansha) |
TASAKA Tetsuya Kyorin University, Faculty of Medicine, Part-time Lecturer, 医学部, 非常勤講師 (70211357)
MATSUZAKI Jun Kyorin University, Faculty of Medicine, Assistant Professor, 医学部, 助手 (80241008)
YONEYAMA Akiko University of Tokyo, Faculty of Medicine, Assistant Professor, 医学部, 助手 (50175684)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1992: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Flow Cytometry / Intracellular pH / Cell Activation / Cell Surface Antigens / レーザー走査型顕微鏡 |
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| Research Abstract |
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 persons
Intracellular 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 antigens
The 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 pHi
the 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 subset
Using 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.
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