Cyanobacterial stabilized phycobilisomes as fluorochromes for extracellular antigen detection by flow cytometry

Phycobilisomes are cyanobacterial photosynthetic energy transfer complexes partly composed of phycobiliproteins, proteins that are widely used as conjugable fluorochromes for flow cytometry. The brightness and photostability of phycobiliproteins suggest that intact phycobilisomes could constitute even brighter probes for fluorescence-based detection systems. Stabilized phycobilisomes have been isolated and the red-excited, far red-emitting Spirulina platensis-derived complex PBXL-3 was accessed as a fluorochrome for flow cytometric immunodetection of surface antigens on immune cells. Although the large size of intact phycobilisomes initially precluded efficient cell surface labeling, the addition of a PEG spacer arm between PBXL-3 and its conjugated avidin molecule (designated PBXL-3L) reduced the steric hindrance associated with the high molecular weight PBXL complex. PBXL-3L increased the surface labeling surface-to-noise ratio and subsequent sensitivity by several-fold over commonly used red-excited fluorochromes such as APC. Interestingly, low power laser sources (including helium-neon and red diode) were particularly efficient at exciting PBXL-3. PBXL-3 was also compatible in with other fluorochromes for multicolor flow cytometry applications. In summary, PBXL-3 was found to possess superior sensitivity and efficiency for flow cytometric immunodetection, particularly with low power laser sources.