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Figure 6: Expression of endogenous FVIII protein in human PBMCs. (a–e) PBMCs were isolated from five unrelated human donors using lymphocyte separation medium and gradient centrifugation. Fixed and permeabilized PBMCs were incubated with anti-FVIII monoclonal antibodies ab41188 and ESH8. Binding of FVIII protein was detected using Alexa Fluor 488 labeled goat anti-mouse IgG secondary antibodies. Each histogram depicts the fluorescence intensity of 10,000 cells labeled with the isotype control IgG2a (blue), ab41188 (red), or ESH8 (orange). (f) Bar and whisker plots show the median fluorescence of all five donors when using IgG2a isotype control antibody and the FVIII-specific antibodies ESH8 and ab41188. On each box, the central mark is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points. The fluorescence is significantly higher when the cells are labeled with the FVIII-specific antibodies ESH8 and ab41188, than when labeled with the isotype control antibody, IgG2a ( and 0.01, resp.).