Open AccessAttachment of A172 human glioblastoma cells affects calcium signalling: A comparison of image cytometry, flow cytometry, and spectrofluorometry
Author(s) -
Szöllösi Jànos,
Feuerstein Burt G.,
Hyun William C.,
Das Manoj K.,
Marton Laurence J.
Publication year - 1991
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990120804
Subject(s) - flow cytometry , cytometry , fluorescence , trypsinization , chemistry , biophysics , calcium , microbiology and biotechnology , biology , biochemistry , physics , optics , trypsin , organic chemistry , enzyme
The intracellular free calcium concentration ([Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.