Model system evaluating fluorescein-labeled microbeads as internal standards to calibrate fluorescence intensity on flow cytometers.
Fluorescence intensity calibration was evaluated in a model system for flow cytometers using commercially available fluorescein-labeled microbeads as internal standards and stabilized fluoresceinated thymus cell nuclei (Fluorotrol) as surrogates for stained mononuclear cells. Spectrophotometrically determined calibration values for the microbeads were used to generate a standard curve that converted green fluorescence histogram channels into molecular equivalents of soluble fluorescein (MESF). In 19 analyses repeated during a single run, the coefficients of variation (CVs) for the derived MESF values on both dimly and brightly stained Fluorotrol populations were less than 2%. In 26 separate determinations over 14 weeks, the CVs of the derived MESF values were less than 3%. The MESF values of the dim and bright Fluorotrol populations derived from the microbead standard curves were both about 50% lower than those determined by direct spectrophotometric analysis of Fluorotrol. The analytical imprecision of fluorescence intensity measurements in this idealized model system has a CV less than 3%, and the analytical inaccuracy shows that calibration in MESF units remains uncertain over about a two-fold range.
Vogt
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Division of Environmental Health Laboratory Sciences Centers for Disease Control, Atlanta, Georgia 30333.
Cross
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Henderson
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Phillips
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D000818:Animals; D002138:Calibration; D002417:Cattle; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D008863:Microspheres; D013950:Thymus Gland / Q000166:cytology*; D014894:Weights and Measures
D005456:Fluorescent Dyes