Application of analytical cytology to current issues in the biomedical sciences

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Flow Cytometry Shared Resource Facility (FCU) initiatives: Because of flow cytometry core responsibilities, these projects are on hold. While users of the flow cytometry core have a broad range of expertise in this technology, we frequently have encountered situations where the design and implementation of their reagent panels is less than optimal. We realized that a paper documenting the proper strategies for selecting both compensation reagents and performing proper flow cytometric compensation for sample validation would benefit both our user base in the IRP as well as the flow cytometry community at large. We undertook a comprehensive analysis of typical compensation reagents, i.e. single color antibody controls using the same reagents in an antibody panels and expanded this to the use of CD8 and CD45 antibodies conjugated to each of the fluorochromes in the panel, and in collaboration with Spherotech Inc., the use of antigen-coated beads as compensation controls. This human-focused study also utilized a large number of antibody panels (12) that ranged from the simple (4-color), to 6- and 8-color, including panels that violated the common flow cytometric rules of antibody-fluorochrome selection, commercial panels, and the Euroflow panels used in the NIH trans-immunology imitative. Data acquisition is completed and an initial review of the results indicates there are a number of instances where the use of the same color antibodies (i.e. CD28 in the panel with use of CD28 as a compensation control) and either CD45 or CD8-matched fluorochrome controls are inappropriate. The results also show major differences in these beads among manufacturers adding to errors in selecting and setting compensation controls.