Abnormal spectra alteration observed in Triton calibration method for measuring [Ca2+]i with fluorescence indicator, fura-2.
We compared two commonly used calibration methods for measuring the concentration of intracellular free calcium ([Ca2+]i) by ratiometric fluorescence dye, fura-2 in mouse neuroblastoma-rat glioma hybrid cells (NG108-15). One calibration method, the Triton method, employs detergent Triton X-100, while the other, the Ionomycin method, uses a calcium-specific ionophore, Ionomycin. In the Triton method, we observed that at excitation 380 nm, the fura-2 fluorescence intensity of steady-state cells abnormally situated beyond the limiting intensity for calibration. By excitation scan, we demonstrated that this abnormality was caused by the change of fura-2 isosbestic points, which in turn was due to cell lysis after the addition of Triton X-100. This problem was resolved in the Ionomycin method by avoidance of cell lysis. Our results showed the correlation between inconsistent isosbestic points and cell lysis. As the basis for [Ca2+]i calibration, the proportionality between the fluorescence intensity and the concentration of dye species was impaired because of inconsistent isosbestic points. This inconsistency can be eliminated by a preliminary experiment of excitation scan to test the feasibility of different calibration methods.
Xu
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Bioelectromagnetic Lab, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100080, China. xutao747@yahoo.com
Yang
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W
Huo
X L
XL
Song
T
T
D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000737:chemistry; D002138:Calibration / Q000592:standards; D002460:Cell Line; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D016257:Fura-2 / Q000032:analysis* / Q000737:chemistry; D051379:Mice; D017830:Octoxynol / Q000737:chemistry*; D051381:Rats; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D017830:Octoxynol; D002118:Calcium; D016257:Fura-2