Fluorescence measurements of free Ca2+ concentration in human erythrocytes using the Ca2+-indicator fura-2.
We report here the use of the fluorescent Ca2+-chelator fura-2 to directly measure free Ca2+ concentration within intact human erythrocytes and the influence of viscosity on the fluorescence of this probe. The bright fluorescence of fura-2 has permitted the use of low concentrations of indicator and cells, thus minimizing the screening effect and the intrinsic fluorescence of haemoglobin. Erythrocytes (10(8) cells/ml) were loaded with 0.5 microM fura-2AM then diluted at 10(7) cells per ml for measurements. The extracellular signal was suppressed by addition of manganese ions just before recording spectra. Under these conditions, a blood sample of 100 microliter was sufficient for analysis. To study the influence of viscosity on fura-2 fluorescence, gelatin and polyvinylpyrrolidone at various concentrations were added to a physiological buffer to perform fura-2-Ca fluorescence standard curves. Fluorescence intensities and the apparent affinity constant for Ca2+ were modified by viscosity. When intra-erythrocytic viscosity was simulated with 21 g/l polyvinylpyrrolidone to obtain a mean viscosity of 14 mPa.s similar to that observed in human erythrocytes, the mean value of free Ca2+ concentration measured in erythrocytes from healthy subjects was 78 +/- 16 nM (mean +/- S.D., n = 29).
David-Dufilho
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M
Département de Pharmacologie, U7 INSERM/UA318 CNRS, Hôpital Necker, Paris, France.
Montenay-Garestier
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T
Devynck
M A
MA
D001572:Benzofurans / Q000378:metabolism; D002118:Calcium / Q000097:blood* / Q000378:metabolism; D004912:Erythrocytes / Q000032:analysis*; D005456:Fluorescent Dyes / Q000032:analysis; D016257:Fura-2 / Q000031:analogs & derivatives*; D006801:Humans; D013050:Spectrometry, Fluorescence / Q000379:methods
D001572:Benzofurans; D005456:Fluorescent Dyes; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2