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Fluorescent determination of chloride in nanoliter samples

García, Néstor H. ; Plato, Craig F. ; Garvin, Jeffrey L. ; García, Néstor H.

Kidney International, January 1999, Vol.55(1), pp.321-325 [Peer Reviewed Journal]

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  • Title:
    Fluorescent determination of chloride in nanoliter samples
  • Author: García, Néstor H. ; Plato, Craig F. ; Garvin, Jeffrey L. ; García, Néstor H.
  • Description: Fluorescent determination of chloride (Cl-) in nanoliter samples. BackgroundMeasurements of Cl- in nanoliter samples, such as those collected during isolated, perfused tubule experiments, have been difficult, somewhat insensitive, and/or require custom-made equipment. We developed a technique using a fluorescent Cl- indicator, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), to make these measurements simple and reliable. MethodsThis is a simple procedure that relies on the selectivity of the dye and the fact that Cl-quenches its fluorescence. To measure millimolar quantities of Cl- in nanoliter samples, we prepared a solution of 0.25 mM SPQ and loaded it into the reservoir of a continuous-flow ultramicrofluorometer, which can be constructed from commercially available components. Samples were injected with a calibrated pipette via an injection port, and the resultant peak fluorescent deflections were recorded. The deflections represent a decrease in fluorescence caused by the quenching effect of the Cl- injected. ResultsThe method yielded a linear response with Cl- concentrations from 5 to 200 mM NaCl. The minimum detectable Cl- concentration was approximately 5 mM. The coefficient of variation between 5 and 200 mM was 1.7%. Resolution, defined as two times the standard error divided by the slope, between 10 and 50 mM and between 50 and 200 mM was 1 mM and 2.6 mM, respectively. Furosemide, diisothiocyanostilbene-2,2′-disulfonic acid and other nonchloride anions (HEPES, HCO3, SO4, and PO4) did not interfere with the assay, whereas 150 mM NaBr resulted in a peak height greater than 150 NaCl. In addition, the ability to measure Cl- did not vary with pH within the physiological range. ConclusionWe developed an easy, accurate, and sensitive method to measure Cl- concentration in small aqueous solution samples.
  • Is Part Of: Kidney International, January 1999, Vol.55(1), pp.321-325
  • Identifier: ISSN: 0085-2538 ; DOI: 10.1046/j.1523-1755.1999.00239.x
  • Subjects: Cotransport ; Furosemide ; Dids ; Bromide ; Kidney ; Thick Ascending Limb
  • Language: English

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