i.paneldevelopmentstrategy.docVIP

I. Panel Development Strategy. We designed our panel using commercially available antibody clones specific for our markers of interest, and selected from available fluorophores to minimize spectral overlap. The most important step for this panel was robust separation of mouse and human leukocytes. We tested several clones specific for mouse and human CD45 (common leukocyte antigen) and tested them against both mouse and human PBMCs to ensure there was minimal cross reactivity (Online Figure 2). As there was virtually no cross reactivity detected with the CD45 specific clones selected, we chose to place them in channels with a high level of spectral overlap, APC and Alexa-700, as those fluorophores would not be attached to the same cell. We then proceeded to assign fluorophores to the other cell surface markers based on availability, staining index and spectral overlap. We also developed this panel with the idea that we may want to use certain anchor markers, such as CD45, CD3, CD4, CD8 and CD19 in supplemental panels to examine other parameters. For example, we’ve used a modified version of this panel to examine NK cell phenotype (Online Figure 4), T cell maturation and chemokine receptor level expression by swapping out the HLA-DR, CD38 and mouse CD45 markers in the FITC, PE and APC channels respectively for other markers of interest (e.g. CD16, CD94, NKG2C and NKG2D for NK Cells, CD45RA and CD27 for T Cell maturation, and CCR5 and CXCR4 for chemokine / HIV co-receptor expression). With this in mind, we chose to place these anchor markers in less common fluorophore channels, such as Alexa700, QDot-605 and Pacific Blue, as there is a greater likely hood of finding new antibody fluorophore combinations commercially available in the more common channels such as PE, FITC, APC and PerCP. This has allowed for greater flexibility when designing additional panels. II. Cross Reference to Related Panels. None To Date. III. Exact Staining Protocol. Mate