APC-conjugated anti-human primary antibodies are far-red fluorescent immunoreagents optimized for flow cytometric detection of human antigens using red laser excitation. Produced by immunizing hosts such as goat, rabbit, or mouse with purified human immunoglobulins or specific human proteins, the antibodies are affinity-purified and conjugated to allophycocyanin, a 105 kDa phycobiliprotein derived from cyanobacteria, primarily Spirulina platensis. APC exhibits excitation/emission maxima at approximately 650/660 nm and is optimally excited by red lasers (633–647 nm) commonly found on modern flow cytometers.
With a molar extinction coefficient of approximately 7.0 × 105 M−1cm−1 and a quantum yield of 0.68, APC is exceptionally bright. Its far-red emission minimizes spectral overlap with FITC (525 nm), PE (578 nm), and PerCP (675 nm), making APC suitable for multicolor flow cytometry panels. Available specificities include anti-human IgG (H+L), anti-human IgG (Fc), isotype-specific reagents targeting IgG1, IgG2, IgG3, IgG4, IgA, and IgM, as well as antibodies against human CD markers such as CD3, CD4, CD8, CD19, CD20, CD25, and CD45. Extensive cross-adsorption against immunoglobulins from mouse, rat, rabbit, and bovine can further reduce nonspecific binding.
Applications
APC conjugates are extensively employed in flow cytometry and fluorescence-activated cell sorting (FACS) for immunophenotyping human leukocyte subsets, including T cells, B cells, NK cells, monocytes, and dendritic cells, in immunology, hematology, and oncology research. Red laser excitation enables their incorporation into multicolor panels alongside fluorophores such as FITC, PE, PerCP, and Pacific Blue™ for comprehensive immune profiling.
In immunofluorescence microscopy and immunohistochemistry, APC can be used for antigen localization through far-red fluorescence detection, which may help reduce interference from tissue autofluorescence. Additional applications include high-content screening, Luminex® multiplex bead-based assays for cytokine profiling, cell proliferation assays using CFSE labeling, and apoptosis detection using annexin V.
Handling and Storage
Typical working dilutions for flow cytometry range from 1:50 to 1:500, depending on the antibody, target antigen, cell type, and experimental conditions. APC-conjugated antibodies should generally be stored at 4°C and protected from light. They should not be frozen unless specifically indicated by the manufacturer. These reagents are intended for research use only and are not intended for diagnostic procedures.