A new standard fluorescence microsphere for quantitative flow cytometry

Area of Science:

• Biotechnology
• Analytical Chemistry
• Immunology

Background:

• Quantitative flow cytometry requires reliable standards for accurate fluorescence measurement.
• Standardized microspheres are essential for calibrating instruments and validating assays.
• Fluorescein isothiocyanate (FITC) is a common fluorophore used in biological labeling.

Purpose of the Study:

• To develop a standard fluorescence microsphere for quantitative flow cytometry.
• To establish a reproducible method for conjugating FITC to microspheres.
• To validate the use of these microspheres for calibrating flow cytometers and quantifying cell-bound molecules.

Main Methods:

• Preparation of uniformly sized plastic microspheres (Micropearl-FT) covalently conjugated with FITC.
• Utilizing alkaline lysis to determine the amount of bound FITC.
• Assessing fluorescence intensity of FITC-conjugated microspheres (FCMS) via flow cytometry.
• Calibrating an Epics V flow cytometer and examining minimum measurable fluorescence intensity.

Main Results:

• The amount of bound FITC was dependent on FITC concentration and incubation time.
• FCMS exhibited linear fluorescence intensity dependence on the amount of bound FITC.
• FCMS fluorescence stability was confirmed for at least 3 months in aqueous solutions.
• The system successfully quantified an average of 4.4 x 10(4) FITC-conjugated OKT3 molecules per T lymphocyte at saturation.

Conclusions:

• A stable and reproducible standard fluorescence microsphere (FCMS) was successfully developed.
• The developed FCMS is suitable for quantitative flow cytometry and instrument calibration.
• This method allows for accurate quantification of specific molecules, such as OKT3, bound to lymphocytes.