Fluorescent determination of chloride in nanoliter samples.
BACKGROUND
Measurements 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.


METHODS
This 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.


RESULTS
The 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.


CONCLUSIONS
We developed an easy, accurate, and sensitive method to measure Cl- concentration in small aqueous solution samples.
García
N H
NH
Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan, USA.
Plato
C F
CF
Garvin
J L
JL
D000818:Animals; D002712:Chlorides / Q000032:analysis*; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007684:Kidney Tubules / Q000378:metabolism; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods / Q000706:statistics & numerical data; D010477:Perfusion; D011806:Quinolinium Compounds; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data
D002712:Chlorides; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium