Figure 4
Figure 4. Colocalization of CD99 and HLA class I. Wild-type Jurkat T cells were fixed, permeabilized, and stained for immunofluorescence microscopy (IFM). (A) CD99 molecules were visualized by indirect fluorescence using the CD99 “O662” mAb followed by a Texas Red–conjugated horse anti–mouse IgG. HLA class I molecules were detected with an FITC-conjugated “W6/32” mAb. (B,C) “O662” or “W6/32” mAbs were used to visualize CD99 or HLA class I molecules, respectively; specific binding was revealed with a Texas Red–conjugated horse anti–mouse IgG. The Golgi apparatus was stained with a FITC-GM130 mAb. (D-F) Intracellular colocalization of CD99 and HLA class I molecules is Golgi dependent. Jurkat cells treated with or without nocodazole (noc.) were fixed, permeabilized, and stained for deconvolution fluorescence microscopy. Nocodazole disturbs the Golgi integrity while the ER remains intact. ER and Golgi compartments were costained either with an anti-Bip/GRP78 mAb followed by a Texas Red–conjugated horse anti–mouse IgG and a FITC-GM130 mAb, respectively. Nocodazole affects the intracellular localization of CD99. The “0662” mAb was used for CD99 labeling, which was detected with a Texas Red–conjugated horse anti–mouse IgG; Golgi was detected with an FITC-conjugated GM130 mAb. CD99 and HLA class I labeling was performed using the “O662” mAb with a Texas Red–conjugated horse anti–mouse IgG and the FITC-“W6/32” mAb, respectively. As a control, cells were treated with cytochalasin B (cyt. B), which disturbs the actin cytoskeleton. (G) Cocapping of CD99 and HLA class I molecules. Jurkat T cells were treated as described in “Methods.” Briefly, cells were incubated for 30 minutes with biotinyled anti–HLA class I “W6/32” mAb followed by a 30-minute incubation with Texas Red–streptavidin. Cells were incubated at 37°C for 60 minutes to allow capping. Labeling for CD99 was then performed by incubation with the “0662” mAb followed by incubation for 30 minutes at 4°C with a Texas Red–conjugated rabbit antimouse antibody. Finally, cells were plated onto PLL-coated slides and fixed before microscopic analysis. Cells in noncapping (NC) and capping (C) conditions are shown in the upper panel and in the lower panel, respectively. No cross-reactivity between the anti–Ig conjugates and the primary antibodies was observed in control cells. (H) Immuno-electron microscopy identifies colocalization of HLA class I and CD99. Consecutive immuno-gold double labeling of Jurkat cells: detection of HLA class I by W632 mAb and protein A coupled to 15-nm gold particles followed by labeling with CD99 (O662 mAb) and protein A coupled to 10-nm gold particles. The arrows point to HLA class I and CD99 colocalization areas.

Colocalization of CD99 and HLA class I. Wild-type Jurkat T cells were fixed, permeabilized, and stained for immunofluorescence microscopy (IFM). (A) CD99 molecules were visualized by indirect fluorescence using the CD99 “O662” mAb followed by a Texas Red–conjugated horse anti–mouse IgG. HLA class I molecules were detected with an FITC-conjugated “W6/32” mAb. (B,C) “O662” or “W6/32” mAbs were used to visualize CD99 or HLA class I molecules, respectively; specific binding was revealed with a Texas Red–conjugated horse anti–mouse IgG. The Golgi apparatus was stained with a FITC-GM130 mAb. (D-F) Intracellular colocalization of CD99 and HLA class I molecules is Golgi dependent. Jurkat cells treated with or without nocodazole (noc.) were fixed, permeabilized, and stained for deconvolution fluorescence microscopy. Nocodazole disturbs the Golgi integrity while the ER remains intact. ER and Golgi compartments were costained either with an anti-Bip/GRP78 mAb followed by a Texas Red–conjugated horse anti–mouse IgG and a FITC-GM130 mAb, respectively. Nocodazole affects the intracellular localization of CD99. The “0662” mAb was used for CD99 labeling, which was detected with a Texas Red–conjugated horse anti–mouse IgG; Golgi was detected with an FITC-conjugated GM130 mAb. CD99 and HLA class I labeling was performed using the “O662” mAb with a Texas Red–conjugated horse anti–mouse IgG and the FITC-“W6/32” mAb, respectively. As a control, cells were treated with cytochalasin B (cyt. B), which disturbs the actin cytoskeleton. (G) Cocapping of CD99 and HLA class I molecules. Jurkat T cells were treated as described in “Methods.” Briefly, cells were incubated for 30 minutes with biotinyled anti–HLA class I “W6/32” mAb followed by a 30-minute incubation with Texas Red–streptavidin. Cells were incubated at 37°C for 60 minutes to allow capping. Labeling for CD99 was then performed by incubation with the “0662” mAb followed by incubation for 30 minutes at 4°C with a Texas Red–conjugated rabbit antimouse antibody. Finally, cells were plated onto PLL-coated slides and fixed before microscopic analysis. Cells in noncapping (NC) and capping (C) conditions are shown in the upper panel and in the lower panel, respectively. No cross-reactivity between the anti–Ig conjugates and the primary antibodies was observed in control cells. (H) Immuno-electron microscopy identifies colocalization of HLA class I and CD99. Consecutive immuno-gold double labeling of Jurkat cells: detection of HLA class I by W632 mAb and protein A coupled to 15-nm gold particles followed by labeling with CD99 (O662 mAb) and protein A coupled to 10-nm gold particles. The arrows point to HLA class I and CD99 colocalization areas.

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