Abstract While flow cytometry continues to be a critical tool for immunologists, one of the more difficult aspects of this practice is panel design. Fully resolving dim populations of interest can be challenging. Low antigen density, fluorescence spillover, available fluorochromes and limited catalog offerings are just a few of the factors that make panel design difficult often leading to sub-optimal resolution. Previously, we have shown that following a basic set of principles can reduce the number of iterations needed to maximize the resolution for populations of interest. Here we show how recent developments, including the BD Horizon™ Guided Panel Solution (GPS) and BD OptiBuild™ reagents, help simplify panel design even further. The BD GPS was designed with the best practices of panel design at its core. By guiding the user through critical steps in this process and flagging potential problem areas in a proposed panel, the GPS minimizes the need for early panel iteration. BD OptiBuild reagents vastly improves the catalog offering of fluorochrome/specificity combinations, thus reducing the need for panel redesign due to limited reagent availability. To demonstrate the utility of these new tools, we walk through panel design for an 8 color assay on both 3 laser (8 parameter) and 5 laser (18 parameter) systems. The analysis of these panels shows that by using these tools, the chances of obtaining a successful panel increase. Additionally, panel design can be made easier and population resolution improved when utilizing a 5 laser system where impacts of spillover and spread can be minimized. Together, using these tools and approaches the goal is to minimize the number of iterations required to develop panels that meet the needs of the investigator.

While UV lasers have been available for many years, their utility has been mainly restricted to DNA/RNA binding fluorochromes (e.g. DAPI, Hoescht 33342) for studying cell cycle or stem cell side populations. The low fluorescence intensity of standard fluorescent dyes, such as AlexaFluor™ 350 or AMCA, have made them impractical for routine immunological flow analyses. Here we report the development of a novel class of highly fluorescent polymeric reporters which are excitable by a UV (355nm) laser. The introduction of these dyes has the potential to dramatically extend the functionality of the UV laser adding up to 6 totally new colors for multi-color cytometric flow studies. The first Brilliant UltraViolet (BUV) polymer has an excitation maximum near 355nm with emission below 405nm. Cross laser excitation is minimal requiring very little compensation into the Brilliant Violet 421 channel. Furthermore these polymers can serve as the basis for developing a wide range of bright, ultraviolet excitable tandem reagents, which span the entire spectrum from UV to the near IR. Data will be presented to demonstrate how the superior staining performance and unique excitation / emission profiles of these new dyes offer a broader pallet of colors for those designing multi-parameter flow panels. Comparative flow performance will be presented as will multicolor staining panels which highlight their compatibility with conventional flow reagents.

Abstract With many cell surface markers expressed at very low levels the ability to identify (and sort) biologically relevant sub-populations by flow cytometry is often limited by the availability of fluorescent reagents sufficiently bright to resolve these markers. In the past year multiple new fluorochromes have been developed which have significantly improved brightness compared to previously available reagents. In particular, BD Horizon™ PE-CF594 and BD Brilliant Violet™ 421 reagents can be 2-5 times brighter than equivalent PE reagents which have typically been the brightest available. These brighter reagents, when used in multicolor analyses, provide multiple benefits including full resolution of small, dim sub-populations resulting in more accurate enumeration and enrichment via sorting. Data will be presented demonstrating this improved resolution using model systems including human T memory and Treg cell sub-populations and murine CD34+ bone marrow cells.

Abstract Bead-based immunoassays enable users to measure multiple proteins in the same sample, significantly increasing the amount of data generated from a single sample. For samples that have significant levels of the cytokines of interest, bead-based immunoassays allow users to get a complete cytokine profile from a single sample in a single experiment. As powerful as bead-based assays are, there are still situations where the levels of cytokines fall below the assay range. Supernatants from un-stimulated cells, samples taken from early time-points in a culture stimulation, and numerous biological samples contain very low levels of some cytokines. Results obtained from these samples, where measurements are taken from below the standard curve range, are often ambiguous, variable, and can be misleading. Advances in technology have enabled the development of more sensitive bead-based immunoassays that can help to more reliably measure samples containing low levels of cytokines. Here, we evaluate the Enhanced Sensitivity (ES) Cytometric Bead Array (CBA) Assays for their use measuring low levels of cytokines in various sample types. Using the ES CBA assays, we were able to detect many cytokines earlier in the response than when using the regular CBA assays. The ES CBA Assays were able to detect cytokines when the regular CBA assays could not. For cytokines that were able to be measured using both the ES and the regular CBA Assays, the results obtained by both assays were comparable.