For determination of LAMP1 and CD63 signal by fluorescence microscopy, unaffected control (HDFs, PCS-201-010; ATCC), affected SLC17A5 c.115C>T (GM08497), and ABE-treated SLC17A5 c.115C>T HDFs were grown on eight-chamber cell culture slides (CellTreat Scientific Products). Cells were fixed with zinc sulfate formalin (Mercedes Scientific), and permeabilized in 100% ethanol before blocking in 1% BSA. Primary antibodies (rabbit against LAMP1; 1:200; Abcam, RRID:AB_775978 ) or (rabbit against CD63; 1:100; R&D Systems; RRID: AB_2927414 ) was applied for 1 h at 37°C before applying secondary antibody (donkey anti-rabbit IgG (H + L)-AF488; 1:500–1,000; Molecular Probes; RRID: AB_2535792 ) for 1 h at 37°C. Slides were mounted using VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories). Images were captured on the Keyence BZ-X800 imaging system at 20× magnification. The exposure time for GFP (AF488) and DAPI are 1 s and 0.01 s, respectively. Quantification of LAMP1 and CD63 signal intensity was performed using CellProfiler33 (link) by establishing a module pipeline which included “IdentifyPrimaryObjects” and “MeasureObjectIntensity.”
Bz x800 imaging system
Manufactured by Keyence
The BZ-X800 imaging system is a high-performance microscope designed for comprehensive observation and analysis of biological samples. It features a high-resolution camera, advanced optics, and intuitive software to capture detailed images and video.
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2 protocols using bz x800 imaging system
1
Fluorescence Microscopy for LAMP1 and CD63 Analysis
2
Canine Osteosarcoma Cell Lines Infected by CAV2-AU-M2
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2D cell culture: The canine oncolytic adenovirus CAV2-AU-M2 was used for the infections. All the canine osteosarcoma cell lines and NCF cells were seeded at a density of 2.5 × 105 cells per well in 1 mL of media in a 12-well plate. After 24 h of incubation at 37 °C, the cells were washed with 1× PBS (Phosphate Buffer Saline, Corning: cat#21-040-CV) and infected with the virus at 4 different multiplicities of infection (MOI; 0, 10, 100, 1000) in low-serum DMEM (2% F.B.S.) with no antibiotics. Cell images were taken every 24 h (0, 24, 48 and 72 h) using a Keyence BZ-X800 imaging system.
3D OS spheroid cultures and infections: A total of 5000 canine osteosarcoma or NCF cells were plated in a low-attachment round-bottom 96-well plate (Corning) in 50 µL of complete media along with 50 µL of ECM (30 µg/mL; Collagen I, Thermo: cat#5056). The plates were centrifuged at 500× g for 5 min. The cells were incubated at 37 °C and routinely monitored for spheroid formation. At 72 h, the cells were infected with CAV2-AU-M2 (MOI; 0, 100 and 1000). Images were captured on day 0, day 4, and day 7.
3D OS spheroid cultures and infections: A total of 5000 canine osteosarcoma or NCF cells were plated in a low-attachment round-bottom 96-well plate (Corning) in 50 µL of complete media along with 50 µL of ECM (30 µg/mL; Collagen I, Thermo: cat#5056). The plates were centrifuged at 500× g for 5 min. The cells were incubated at 37 °C and routinely monitored for spheroid formation. At 72 h, the cells were infected with CAV2-AU-M2 (MOI; 0, 100 and 1000). Images were captured on day 0, day 4, and day 7.
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