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Fluorescence intensity calibration for immunophenotyping by flow cytometry
R F Vogt1, W E Whitfield, L O Henderson
1National Diabetes Laboratory, Division of Laboratory Sciences, Atlanta, Georgia, 30340, USA.
Methods (San Diego, Calif.)
|June 30, 2000
Summary
Calibrating fluorescence intensity (FI) using molecules of equivalent soluble fluorochrome (MESF) provides accurate receptor counts. This standardization enhances flow cytometry data comparability across labs and experiments.
Area of Science:
- Immunology
- Biotechnology
- Analytical Chemistry
Background:
- Flow cytometry commonly uses arbitrary fluorescence intensity (FI) for phenotype classification.
- Accurate quantification of fluorescent conjugates and stained receptors is challenging with relative FI values.
Purpose of the Study:
- To establish a standardized method for quantifying fluorescence intensity in flow cytometry.
- To enable accurate measurement of receptor expression using stoichiometric units.
Main Methods:
- Calibration of fluorescence intensity (FI) to molecules of equivalent soluble fluorochrome (MESF).
- Assessment of antibody-binding capacity (ABC) values through binding chemistry analysis.
Main Results:
- FI can be expressed in stoichiometric MESF units for precise measurements.
- Calibration allows for quality control and comparable analysis across different settings.
- Numeric assessment of ABC values and actual receptor numbers on cells is achievable.
Conclusions:
- Standardized MESF measurements improve the reliability and comparability of flow cytometry data.
- Accurate receptor quantification is possible through careful calibration and binding chemistry assessment.
- This approach facilitates robust quality control and inter-laboratory data consistency.