CD81 neither regulates β1-integrin affinity nor avidity, but it organizes integrin cluster distribution in DCs. (A) Flow cytometric analysis shows that a constitutive CD29 epitope is not altered after RNAi of CD81 (left panel). AB indicates antibody. Furthermore, β1 integrin affinity is not abrogated after RNAi of CD81, as shown by flow cytometry of a β1 integrin activation epitope detected by a monoclonal antibody HUTS-4 (right panel). (B) Immunofluorescence staining of β1 integrins (CD29) in LPS-stimulated CD81 RNAi knockdown Mo-DCs and control cells, respectively. The fluorescence staining images are confocal images (cLSM), focused to the basal plasma membrane of the respective cells. DIC indicates differential interference contrast. (C) Images generated with TIRF microscopy allow the visualization of individual CD29 clusters within the basal plasma membrane with high resolution. Individual CD29 cluster size was measured in central (c) as well as peripheral (p) positions of the basal plasma membrane (PM) with the help of autocorrelation analysis by the use of ImageJ software (bottom panel). The cells were adhered to 2-dimensional fibronectin-coated surfaces (5 μg/cm²). Bars represent 10 and 1 μm (magnified detail), respectively. Epifl. means epifluorescence image of corresponding TIRFM Image (C).