Fluorescent immunophenotyping using the tandem conjugate PerCP-Cy5.5 on the FACSVantage.
The tandem conjugate PerCP-Cy5.5 (developed by Becton-Dickinson Biosciences) can be excited with a standard 488 nm laser and emits in the far red at a slightly longer wavelength than PE-Cy5 (approximately 710 nm). This emission wavelength makes PerCP-Cy5.5 a potentially useful fluorochrome for multicolor analysis with FITC, PE and other fluorochromes. Its tandem structure may make it more photostable than PerCP alone, which generally photobleaches rapidly with more powerful water-cooled gas lasers.
Methods. Normal human PBMCs were isolated from apheresis samples, RBC-depleted with over Ficoll-Hypaque and labeled with PerCP-Cy5.5 conjugated to antibody against the human B lineage antigens CD19 and CD20 at the concentration recommended by the manufacturer. For comparison purposed, cells were labeled in parallel with PerCP--anti-human CD20 (BD Biosciences). Cells were analyzed on both a FACSCalibur equipped with an air-cooled argon-ion laser emitting at 488 nm at 15 mW, and a FACSVantage SE equipped with a water-cooled argon-ion lase emitting at 30 to 150 mW. On the FACSCalibur PerCP-Cy5.5 was detected through a 650 LP emitting dichroic; on the FACSVantage SE, PerCP-Cy5.5 was detected through narrow bandpass filters ranging from 660/20 nm to 710/20 nm, as well as a 650 LP.
(Below). Detection of CD19 and CD20 expression on human PBMCs with PerCP-Cy5.5 (left and middle column) and CD20 with PerCP (left column) using a FACSCalibur (top row) or FACSVantage SE with several emission optics (remaining rows). PerCP-Cy5.5 was nearly undetectable at higher laser powers.
(Below). Detection of CD19 and CD20 expression on human PBMCs with PerCP-Cy5.5 (left and middle column) and CD20 expression with PerCP (right column) using lower laser power and different emission optics. Reduction of laser power to 50 and 30 mW (top two rows) increased detectability of both PerCP-Cy5.5 and PerCP somewhat, but failed to resolve CD19 or CD20-positive populations with PerCP-Cy5.5. Use of narrow bandpass optics for PerCP (third and fourth row) and PerCP-Cy5.5 (fifth and sixth row) did not resolve the problem.
PerCP-Cy5.5 was found to be quite bright on the FACSCalibur, similar to PerCP. However, the fluorochrome was not useful on the FACSVantage at a range of laser power levels, despite emission filters both optimal for the fluorochrome and similar to the FACSCalibur. The inability to detect PerCP-Cy5.5 fluorescence even at lower laser powers (where PerCP was dim but still useable) suggests that a mechanism other than photobleaching may be reducing its effective fluorescence on stream-in-air sorters. Use of both PerCP-and PerCP-Cy5.5 should be limited to benchtop instruments, where they constitute bright and useful fluorescent markers.