Cluster of Differentiation 86 (CD86) is a cell surface marker present on many types of immune cells, including dendritic cells (DC) and their surrogates. CD86 is responsible for T-cell activation as part of the immune response system. As such, measuring CD86 expression in dendritic cell surrogates is critical for several regulatory assays involved in assessing potential immune-sensitizing agents. Consistently measuring CD86 expression can be difficult, and variability in expression levels can be observed trial-to-trial. Previous work has identified some potential solutions, such as using sodium azide as an additive in flow cytometry wash buffer, as well as using cold temperatures when handling post-treatment cells.
Another important aspect when measuring CD86 expression via flow cytometry is to effectively block non-specific binding of the anti-CD86 antibody. A common method employed is to add a small amount of bovine serum (BSA) to the washing buffer used for cell harvesting. The added protein binds to the Fc receptors located on the cell surface, and helps reduce the signal-to-noise ratio. Without effective blocking, the antibody may bind to the Fc receptors, artificially increasing the measured expression of the target cell surface marker.
To identify an effective blocking concentration of BSA in wash buffer, a simple experiment was designed to test three different concentrations: 0.5%, 0.75%, and 1% (w/v). The BSA was sourced from EMD Millipore and was mixed into calcium- and magnesium-free phosphate buffered saline (CMF-PBS) sourced from Gibco. Samples of DC surrogate cells were seeded at a density of 2.0x10^5 cells/mL and washed with an equal volume of wash buffer. The samples were centrifuged at 300 times gravity for five minutes at a temperature of four degrees Celcius. Following centrifugation, the supernatant was aspirated and washed a second time with one milliliter of wash buffer. The cells were once again centrifuged and aspirated, and then finally resuspended in fifty microliters of wash buffer. The samples were stained with FITC-tagged anti-CD86 antibody, sourced from BD Pharmigen, at a previously determined titration (two microliters). A separate sample was concurrently stained with anti-IgG isotype of the same fluorescent marker. The staining solution was allowed to incubate in the dark, in a four degree Celcius refrigerator for thirty minutes on a plate shaker. After the staining incubation, the cells were washed once more with wash buffer and centrifuged as before. The cells were finally resuspended in two-hundred microliters of wash buffer and analyzed by flow cytometry.
The cell population was gated to remove debris. Using the isotype sample, a sub-plot of the cell population was quad-gated for PE and FITC. The isotype cell population was gated to be in quadrant four, with cells appearing in quadrants two and three corresponding to an increase in CD86 expression. The percent of CD86-positive cells compared to the total cell population was recorded for each sample.