The possible pitfalls of the measurement of intracellular calcium concentration of lymphocytes with the fluorescent indicator quin2.
The measurement of intracellular calcium concentration of small cells became feasible and relatively easily accomplishable following the development of the fluorescent calcium chelating dye, quin2, by R. Y. Tsien and his co-workers. The present paper gives an experimental survey of the major possible pitfalls of the method such as: 1) the improper choosing of the concentration of quin2/AM, 2) the misinterpretation of the calibration procedure, 3) the accidental heavy metal traces in the medium, and 4) the uneven distribution of quin2 among the individual cells or within a certain cell. We report some original data on: 1) the distorting effect of heavy metal ions on the measurement and the use of chelex 100 resin to eliminate heavy metal traces from the mediums, 2) the negligible contribution of dead cells to the overall fluorescence signal demonstrated by flow-cytometry, and 3) fluorescence polarization of quin2 in lymphocytes.
D000634:Aminoquinolines; D000818:Animals; D002118:Calcium / Q000032:analysis*; D002470:Cell Survival; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007424:Intracellular Fluid / Q000032:analysis; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D011817:Rabbits; D013601:T-Lymphocytes / Q000032:analysis*
D000634:Aminoquinolines; D005456:Fluorescent Dyes; C035738:Quin2-acetoxymethyl ester; D002118:Calcium