Pacific Blue™ conjugated anti-horse primary antibodies are violet-excitable fluorescent immunoreagents designed for high-sensitivity detection in multicolor flow cytometry and fluorescence microscopy. Produced by immunizing hosts such as goat or rabbit with purified horse immunoglobulins, the antibodies are affinity-purified and covalently conjugated to Pacific Blue™, a small-molecule coumarin-based fluorophore. Pacific Blue™ exhibits excitation/emission maxima at approximately 401–410/452–455 nm and is optimally excited by the 405 nm violet laser line commonly available on modern flow cytometers.
The fluorophore's low phenol pKa (3.7) ensures stable fluorescence at physiological pH (7.0–7.4), minimizing pH-dependent signal fluctuations in live-cell assays. With a molar extinction coefficient of 46,000 cm−1M−1 and a quantum yield of 0.78–0.91, Pacific Blue™ provides moderate-to-high brightness suitable for detecting low-abundance equine antigens. The blue emission (approximately 455 nm) exhibits minimal spectral overlap with green fluorophores such as FITC and PE, facilitating multicolor panel design. However, significant spectral overlap with Brilliant Violet 421™ (BV421) generally precludes their combined use in conventional flow cytometry; resolution may be possible with spectral flow cytometry platforms employing mathematical unmixing. Available specificities include anti-IgG (H+L), anti-IgG subclass, and anti-IgM reagents.
Applications
Pacific Blue™ conjugates are primarily employed in flow cytometry and fluorescence-activated cell sorting (FACS) for immunophenotyping equine leukocytes and detecting cell-surface markers such as CD4, CD8, and MHC class II. Violet laser excitation enables simultaneous detection with other fluorophores in high-parameter multicolor panels. In immunofluorescence microscopy and immunohistochemistry, the fluorophore's photostability supports imaging with reduced photobleaching. Additional applications include high-content screening, fluorescent Western blotting, and multiplex bead-based assays.
Typical working dilutions range from 1:50 to 1:500 for flow cytometry. Antibody conjugates should be stored at 4°C, protected from light, and should not be frozen. These reagents are intended for research use only and are not for diagnostic procedures.