U2OS, 4T1 and A549 cells were seeded 18 h prior to treatment at a density of 50,000 cells per confocal chamber. Then, cells were washed twice with PBS and incubated with culturing media containing Rhodamine-labeled pCQ (F-pCQ10.0) for 24 h. Rhodamine-labeled pHPMA (F-pHPMA) was used as a control either alone or co-incubated with pCQ16.7 or HCQ. Cells were washed five times with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min. Fixed cells were washed four times with PBS and nuclei were stained with 1 μM Hoechst 33258 solution for 30 min before imaging. The images were obtained using Zeiss 800 Airyscan Microscope coupled with 63x oil objective and z-axis motor.
800 airyscan microscope
Manufactured by Zeiss
The Zeiss 800 Airyscan Microscope is a high-resolution confocal imaging system. It utilizes an Airyscan detector to capture high-quality, high-resolution images. The microscope is designed for advanced imaging applications.
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Lab products found in correlation
24 well glass bottom plate, by Cellvis (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Image express micro confocal high content imaging system, by Molecular Devices (2 mentions)
Alexa fluor 488 647 conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Rabbit anti phospho upf1 antibody, by Merck Group (2 mentions)
4 protocols using 800 airyscan microscope
1
Cellular Uptake of Rhodamine-Labeled Compounds
2
Immunofluorescence Staining of HEK293T and iPSNs
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HEK293T cells were fixed and stained as previously described (Coyne et al., 2020 (link)) using Rabbit anti-phospho-UPF1 antibody (Millipore Sigma) at a 1:250 dilution and AlexaFluor 488 conjugated Goat anti-Rabbit secondary antibody (Thermo Fisher Scientific) at a 1:1000 dilution in a 24-well glass-bottom plate (Cellvis). 9 sites in each well were imaged using a 20x objective on an ImageExpress Micro Confocal High-Content Imaging System (Molecular Devices).
iPSNs were fixed with 4% (v/v) para-formaldehyde in PBS for 10 min, permeabilized in 0.2% (v/v) Triton X-100 for 10 min, blocked in 1% bovine serum albumin and 2% goat serum for 1h, incubated with primary antibodies overnight at 4°C, washed with PBS, and finally incubated with Alexa Fluor 488/647 conjugated secondary antibodies (ThermoFisher Scientific). Cells were imaged with a Zeiss 800 Airyscan microscope.
iPSNs were fixed with 4% (v/v) para-formaldehyde in PBS for 10 min, permeabilized in 0.2% (v/v) Triton X-100 for 10 min, blocked in 1% bovine serum albumin and 2% goat serum for 1h, incubated with primary antibodies overnight at 4°C, washed with PBS, and finally incubated with Alexa Fluor 488/647 conjugated secondary antibodies (ThermoFisher Scientific). Cells were imaged with a Zeiss 800 Airyscan microscope.
3
CXCR4 Trafficking Assay with HCQ
4
Immunofluorescence Staining of HEK293T and iPSNs
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