Immunofluorescent analysis of antibody-induced capping of CD28. Jurkat T cells expressing CD28 were incubated in culture medium with 2 μg/mL divalent CD28.3 IgG antibody (A-B) or 2 μg/mL CD28.3 Fab fragments, with 2 μg/mL scFv28.3-HaaT (E) or with 50% Cos cell supernatant containing approximately 4 μg/mL scFv28.3 (D). Cells were incubated for 1 hour at 0°C (A) or at 37°C (B-E), washed in ice-cooled PBS containing NaN₃, and centrifuged onto microscope slides. After fixation with 0.5% paraformaldehyde (PFA), CD28 molecules were revealed using FITC-labeled CD28.6, a mAb reacting against an epitope on CD28 other than CD28.3.¹¹  Slides were observed by fluorescence microscopy, and magnification is × 63 for all panels.