Cell surface antigens can be detected by monoclonal antibodies conjugated either directly or indirectly to fluorochromes. When appropriately chosen, up to five fluorochromes (surface antigens) may be detected simultaneously with the FACS Vantage (a maximum of four on the FACSCalibur, and three on the FACScan). At present, the FACSAria can detect eleven fluorochomes; the remaining two fluorescence detectors await suitable fluorochromes to be developed to support an amazing 13-color capability.
An example from Dr. Fayth Yoshimura's research on MCF-7 virus-induced leukemia (see below for reference). Murine thymocytes were simultaneously labeled for four surface markers 514-FITC (detects MCF-7 virus on the surface of infected cells), CD3-PE (T cells), CD4-CyChrome* (T helper), and CD8-Cascade Blue* (T suppressor) to determine which T cell subset(s) became infected by the MCF-7 virus. Cells expressing virus (514+, green in the histogram above) were selected (gated) and percentages of T cell subsets were determined using Paint-A-Gate PRO* software. Since primarily immature T cells reside in the thymus, the vast majority of virus-infected cells have CD3+CD4+CD8+ phenotype (black dots in the dot plots above). Only 1.02% of infected cells were mature T helper (CD3+CD4+CD8-, cyan dots), and 0.13% were mature T suppressor (CD3+CD4-CD8+, magenta dots).
*CyChrome and Paint-A-Gate PRO are trademarks of BD Biosciences. Cascade Blue is a registered trademark of Molecular Probes.
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