ATTACHMENT OF A172 HUMAN GLIOBLASTOMA CELLS AFFECTS CALCIUM SIGNALING - A COMPARISON OF IMAGE CYTOMETRY, FLOW-CYTOMETRY, AND SPECTROFLUOROMETRY

被引:18
作者
SZOLLOSI, J
FEUERSTEIN, BG
HYUN, WC
DAS, MK
MARTON, LJ
机构
[1] DEBRECEN UNIV MED, DEPT BIOCHEM & BIOPHYS, H-4012 DEBRECEN, HUNGARY
[2] UNIV CALIF SAN FRANCISCO, SCH MED, DEPT NEUROL SURG, BRAIN TUMOR RES CTR, SAN FRANCISCO, CA 94143 USA
[3] UNIV CALIF SAN FRANCISCO, SCH MED, DEPT LAB MED, SAN FRANCISCO, CA 94143 USA
[4] UNIV CALIF SAN FRANCISCO, SCH MED, DEPT PEDIAT, SAN FRANCISCO, CA 94143 USA
来源
CYTOMETRY | 1991年 / 12卷 / 08期
关键词
CYTOSOLIC FREE CALCIUM CONCENTRATION; INDO-1; MONOLAYER; PLATELET DERIVED GROWTH FACTOR;
D O I
10.1002/cyto.990120804
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The intracellular free calcium concentration ([Ca2+]i) of indo-1 loaded A172 human glioblastoma cells stimulated by platelet-derived growth factor (PDGF) was studied in cell suspensions by flow cytometry and spectrofluorometry and in confluent monolayers by laser image cytometry and spectrofluorometry. With all three techniques, the percentage of responsive cells, peak [Ca2+]i, and the duration of response were directly related, and the delay time was inversely related to PDGF dose. The maximum response occurred at a PDGF concentration of about 20 ng/ml. Basal and peak [Ca2+]i did not differ significantly from method to method even though different calibration procedures were used. Cells in suspension monitored by both spectrofluorometry and flow cytometry displayed significantly shorter calcium responses than attached cells. This did not appear to be a direct effect of trypsinization. Spectral analysis of indo-1 in cytoplasm, 40% glycerol, and aqueous solutions showed significant differences in the isosbestic point and quantum efficiency. Calibration of [Ca2+]i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.
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页码:707 / 716
页数:10
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