CD99 is recruited into the IS and lipid rafts. (A) DDAO-SE pre-labeled Jurkat T cells were incubated with SEB-loaded Raji B cells. Cells were then plated onto a cover slip, fixed, stained with anti-CD99 mAb followed by Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies, TRITC-phalloidin for F-actin and Hoechst 33258 for the nucleus. The stained cells were visualized by confocal fluorescence microscopy. The green and yellow images represent the localization and distribution of CD99 and F-actin, respectively. The nuclei and Jurkat cells are shown in blue and red, respectively. (B) Jurkat cells were homogenized to isolate cell membranes, which were then solubilized in lipid raft preserving detergent 1% Brij-98 or lipid raft disrupting detergent 1% Lauryl maltoside (LM) and subjected to gel filtration on Sepharose 4B. The obtained fractions were analyzed by Western immunoblotting using anti-CD99 and anti-Lck mAbs.