Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2.
The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.
Komada
H
H
Department of Pathology, Mie University School of Medicine, Japan.
Nakabayashi
H
H
Nakano
H
H
Hara
M
M
Yoshida
T
T
Takanari
H
H
Izutsu
K
K
D000634:Aminoquinolines / Q000592:standards; D001572:Benzofurans / Q000592:standards; D002118:Calcium / Q000378:metabolism*; D002453:Cell Cycle; D003208:Concanavalin A / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004987:Ethers / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000592:standards; D005470:Fluorometry; D016257:Fura-2; D006801:Humans; D015759:Ionomycin; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000634:Aminoquinolines; D001572:Benzofurans; D004987:Ethers; D005456:Fluorescent Dyes; D013205:Staphylococcal Protein A; D003208:Concanavalin A; D015759:Ionomycin; D004247:DNA; D013755:Tetradecanoylphorbol Acetate; C035739:Quin2; D002118:Calcium; D016257:Fura-2