Confocal microscopy for cell binding and internalization

In one day before confocal experiment, 4 × 10⁴ cell per well of two cell lines, 293 T and Env-transfected-293T cells, were seeded on a multiple-chamber slide (Nalge Nunc International). The next day, in order to block the IT internalization, the media of two control wells were replaced with PBS/BSA/0.01% sodium azide (PBA) 400 µl per well for 45 min in RT. Then, cells were treated by 1 µg Ab/IT in 500 µl PBS or PBA, incubated 60 min in RT. The antibodies were 7B2-Alexa488, 7B2-PAC-Alexa488 and 7B2-RAC-Alexa488 in the presence of 1 µg sCD4-IgG2). The controls were chRAC18 and sCD4-IgG2-Alexa488. One well was left without adding antibody. BFA-bodipy (250 ng/ml) was added to cells to demonstrate rapid accumulation of toxins in Golgi and ER. BFA-bodipy was tested to determine that at the concentration used there was minimal effect of the BFA on the cytotoxicity of ITs³². After 2X washing with PBA, the cells were fixed in 2% paraformaldehyde/PBS in 45 min. We took out the solution, and removed the slide box, added one drop SlowFade Gold Mountant DAPI (Life Technologies) and covered it by slide glass. Images were obtained with an inverted Zeiss LSM 510 microscope, a 63 × 1.4 NA oil-immersion objective, and Zeiss LSM software. For each cell, a Z-stack with 1 µm steps was imaged.