SCCVII-rPDGFRβ cells were seeded in a glass bottom µ-slide 8-well chambered coverslip (Ibidi, Gräfelfing, Germany) (approximately 104 cells per well) and allowed to adhere overnight at 37 °C, 5% CO2 in a humidified incubator. The next day, the cells were transferred the TokaiHit incubation chamber of the microscope to keep the cells at 37 °C. At the starting point of the timelapse-experiment, 10 nM sdAb conjugated to maleimide pHrodo™ Red (Thermo Fisher Scientific, Bleiswijk, The Netherlands) was added to the cells. Live-cell imaging was performed using a using a Nikon Eclipse Ti confocal spinning disc microscope equipped with Perfect Focus System and a 60× oil objective. Metamorf software was used to make a time-lapse video (picture every 15 sec with 250 ms exposure time). Acquired files were background corrected and time stamped with Fiji/ImageJ software.
Eclipse ti confocal spinning disc microscope
Manufactured by Nikon
The Eclipse Ti confocal spinning disc microscope is a high-performance imaging system designed for advanced microscopy applications. It features a spinning disc confocal design that allows for rapid, high-resolution imaging of live cells and samples. The microscope is capable of capturing real-time, high-speed images with minimal phototoxicity and photobleaching.
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Lab products found in correlation
2 protocols using eclipse ti confocal spinning disc microscope
1
Live-cell imaging of sdAb trafficking
2
Reconstituting COPII Vesicle Formation
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COPII proteins were purified from yeast strains as described before. Sar1 was chemically labeled with Alexa-647 maleimide (ratio 1:5 with Molecular Probes product no. A20347) and Sec13/31 was labeled similarly with TFP-Alexa 488 as described here [30 (link)] (Life technology product no. A37563). In a 50 μL microfluidic chamber (ibidi μ-Slide), 10 μL avidin (Sigma-Aldrich A9275) at 1 mg/mL were pipetted. After 5 min, avidin was washed 5 times with 40μL B88 (469 mOsm). After this, 10 μL of GUVs were pipetted, letting them sit 2 min on the chamber floor. Immediately after, 10μL of 4 mg/mL casein (Sigma-Aldrich C6905-1G) was added to block GUVs binding. No less than 5 washes with B88 469 mOsm were done. The COPII proteins mix was Sar1-Alexa647 at 0.05 μg/μL, Sec23/24 at 0.06 μg/μL, Sec13/31-Alexa488 at 0.09 μg/μL. Next 1 mM GMP-PNP was added and 4 mM EDTA was added to promote Sar1 nucleotide exchange. The protein mix was incubated 5 min at room temperature < 23°C before pipetting it into the microfluidic chamber (ibidi μ-Slide) with GUVs.
Microscopy was performed using a Nikon Eclipse Ti confocal spinning disc microscope with 100x magnification. Images were analyzed with Fiji.
Microscopy was performed using a Nikon Eclipse Ti confocal spinning disc microscope with 100x magnification. Images were analyzed with Fiji.
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