Detection of cell-surface antigens using antibody-conjugated fluorospheres (ACF): application for six-color...

Area of Science:

• Immunology
• Biotechnology
• Analytical Chemistry

Background:

• Multicolor flow cytometry is crucial for immunophenotyping but limited by fluorochrome spectral overlap.
• Existing protocols struggle with simultaneous detection beyond four colors due to spectral interference.
• Novel fluorosphere technologies are needed to enhance multicolor fluorescence capabilities.

Purpose of the Study:

• To introduce Antibody-conjugated fluorospheres (ACF) as a versatile tool for multicolor flow cytometry.
• To demonstrate the utility of ACF in overcoming spectral limitations in immunophenotyping.
• To enable the simultaneous detection of six cell-surface antigens on murine leukocytes.

Main Methods:

• Development and application of Antibody-conjugated fluorospheres (ACF) with unique excitation/emission spectra.
• Utilized SKY BLUE fluorospheres conjugated to CD11b monoclonal antibody.
• Combined ACF with multiple fluorochrome-conjugated monoclonal antibodies (IAd-FITC, L3T4-PE, LYT2-APC, THY1.2-biotin, B220-RED613) for six-color analysis.

Main Results:

• Successfully resolved all fluorescence signals in a six-color immunophenotyping assay.
• Demonstrated equivalent percentages of antigen-positive cells compared to five- and single-color analyses.
• Validated the accuracy and reliability of ACF for complex cell surface antigen detection.

Conclusions:

• Antibody-conjugated fluorospheres (ACF) significantly enhance multicolor flow cytometry protocols.
• ACF provide a versatile solution to spectral overlap challenges, enabling higher-order multiplexing.
• This approach facilitates accurate and simultaneous detection of multiple cell-surface antigens in complex cell populations.