Lsm5 meta

To detect the antigen specific localization of IR700 conjugates, fluorescence microscopy was performed (IX61 or IX81; Olympus America, Melville, NY, USA). Ten thousand cells were seeded on cover-glass-bottomed dishes and incubated for 24 hr. APC was then added to the culture medium at 10 μg/mL and incubated at 37°C for 6 hr. The cells were then washed with PBS; Propidium Iodide (PI)(1:2000)(Life Technologies) and Cytox Blue (1:500)(Life Technologies), were used to detect dead cells. These were added to the media 30 min before observation. The cells were then exposed to NIR light and serial images were obtained. The filter was set to detect IR700 fluorescence with a 590–650 nm excitation filter, and a 665–740 nm band pass emission filter.
3D reconstructions of the spheroids were obtained with a confocal laser microscope (LSM5 meta, Carl Zeiss, Jena, Germany) after incubation for 30 min with Hoechst 33342 (1:500)(Life Technologies). Sections of spheroids were first fixed with 3.7% formaldehyde in PBS for 10 min at room temperature followed by embedding with OCT (SAKURA, Tokyo, Japan). Then, they were frozen at −80°C, cryotomed to obtain 10μm sections (LEICA CM3050 S, Leica microsystems, Wetzlar, Germany). Analysis of the images was performed with ImageJ software (http://rsb.info.nih.gov/ij/).

To detect the antigen specific localization of each conjugate, fluorescence microscopy was performed with a confocal laser microscope (LSM5 meta, Carl Zeiss, Jena, Germany). Ten thousand cells were seeded on cover-glass-bottomed dishes and incubated for 24 h. Each mAb-dye conjugate was then added to the culture medium at 10 µg/mL and incubated at 4 °C (on ice) for 1 h. After 1 h incubation on ice, the media with conjugates was changed to new media (containing no conjugates), then cells were observed after 6 h incubation at 37 °C.
Following 1 h incubation with each conjugate and confirmation of fluorescence signal, cells were washed with medium, and new medium was added and incubated for 3 days. Then, cells were observed with microscopy.
Analysis of the images was performed with ZEN software (Carl Zeiss).

To detect the antigen specific localization of IR700 conjugates, fluorescence microscopy was performed (IX61 or IX81; Olympus America, Melville, NY, USA). Ten thousand cells were seeded on cover-glass-bottomed dishes and incubated for 24 hr. Cet-IR700 or pan-IR700 was then added to the culture medium at 10 μg/mL and incubated at 37°C. The cells were then was hed with PBS; Propidium Iodide (PI) (1:2000) (Life Technologies) and Lyso Tracker Red DND-99 (lysotracker, final 75 nM; Life Technologies), were used to detect dead cells, and acidic organelles, respectively (Raben et al., 2009 ; Smith et al., 2012 (link)). PI was added into the media 30 min before PIT. The cells were then exposed to NIR light (2J/cm²) and serial images were obtained. The filter was set to detect IR700 fluorescence with a 590–650 nm excitation filter, and a 665–740 nm band pass emission filter.
Immunostaining was performed as previously described (Sato et al., 2011 (link)); briefly, the cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature followed by permeabilization for 10 min with 0.2% Triton X-100 containing 2 mg/mL BSA. Alexa-fluor488 Phalloidin (Life Technologies) was used to detect actin. The fixed samples were observed with a confocal laser microscope (LSM5 meta, Carl Zeiss, Jena, Germany). Analysis of the images was performed with ImageJ software (http://rsb.info.nih.gov/ij/).

To detect the antigen specific localization of each conjugate, fluorescence microscopy was performed with a confocal laser scanning microscope (LSM5 meta, Carl Zeiss, Jena, Germany). Ten thousand cells were seeded on coverglass-bottomed dishes and incubated for 24 h. Each mAb-dye conjugate or free dye was then added to the culture medium at 10 μg/mL or 0.5 μM, respectively, and incubated at 4 °C (on ice) for 1 h. The media containing conjugates or dyes was changed to new media (containing no conjugates/dyes) and cells observed after a 6 h incubation at 37 °C.
Alternatively, cells were incubated for 1 h with each conjugate or free dye and the presence of a fluorescence signal confirmed. Cells were then washed with medium, new medium (containing no conjugates/dyes) added, and incubated for 3 days, at which time cells were observed by microscopy. Image analysis was performed with ZEN software (Carl Zeiss).

To detect the antigen specific localization of each conjugate, fluorescence microscopy was performed with a confocal laser scanning microscope (LSM5 meta, Carl Zeiss, Jena, Germany). Ten thousand cells were seeded on coverglass-bottomed dishes and incubated for 24 h. Each mAb-dye conjugate or free dye was then added to the culture medium at 10 μg/mL or 0.5 μM, respectively, and incubated at 4 °C (on ice) for 1 h. The media containing conjugates or dyes was changed to new media (containing no conjugates/dyes) and cells observed after a 6 h incubation at 37 °C.
Alternatively, cells were incubated for 1 h with each conjugate or free dye and the presence of a fluorescence signal confirmed. Cells were then washed with medium, new medium (containing no conjugates/dyes) added, and incubated for 3 days, at which time cells were observed by microscopy. Image analysis was performed with ZEN software (Carl Zeiss).