For time-lapse phase-contrast imaging, HeLa cells were plated on glass bottom 12-well plates (MatTek) and put in an open chamber (Life Imaging) equilibrated in 5% CO2 and maintained at 37 °C. Time-lapse sequences were recorded every 10 min for 48 h using an inverted NikonEclipse TiE microscope with a ×20 0.45 NA Plan Fluor ELWD controlled by Metamorph software (Universal Imaging). For time-lapse fluorescent microscopy, images were acquired using an inverted Eclipse TiE Nikon microscope equipped with a CSU-X1 spinning disk confocal scanning unit (Yokogawa) and with a EMCCD Camera (Evolve 512 Delta, Photometrics). Images were acquired with a ×60 1.4 NA PL-APO VC and MetaMorph software (MDS).
Glass bottom 12 well plates
Manufactured by MatTek
Glass bottom 12-well plates are a type of laboratory equipment used for cell culture applications. These plates have a transparent glass surface at the bottom of each well, which allows for direct visualization and imaging of cells under a microscope. The glass bottom provides a high-quality optical interface for improved imaging performance compared to standard plastic-bottom plates.
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Lab products found in correlation
Csu x1 spinning disk confocal scanning unit, by Yokogawa (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
U2os cell, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Capan 1 cells, by American Type Culture Collection (1 mentions)
Eclipse ti e microscope, by Nikon (1 mentions)
Metamorph software, by Universal Imaging (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti tom20 rabbit sc11415, by Santa Cruz Biotechnology (1 mentions)
6 protocols using glass bottom 12 well plates
1
Time-Lapse Imaging of HeLa Cells
2
Live-cell Imaging of Cell Cycle
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Procedures for EdU analyses and fluorescence microscopy for cells and FFPE-fixed tumor samples are described in the Supplemental Material . All primary and secondary antibodies, along with their dilutions (in PBS + 0.1%Triton X-100, 1% BSA), are shown in Supplemental Table S7 .
For live-cell imaging of the FUCCI system, cells were seeded on glass-bottom 12-well plates (MatTek) for 2 d in the presence of 1 µg/mL doxycycline, and the media was changed 1 h before imaging. Cells were monitored in a humid tissue culture chamber at 37°C with CO2, and multipoint images were taken using a Nikon Elipse Ti fluorescent microscope with a Plan Fluor 10×/0.3 objective, ORCA-R2 camera, and MetaMorph software. Color composite time-lapse images were exported as movies using MetaMorph and viewed and evaluated using Fiji (ImageJ) software.
For live-cell imaging of the FUCCI system, cells were seeded on glass-bottom 12-well plates (MatTek) for 2 d in the presence of 1 µg/mL doxycycline, and the media was changed 1 h before imaging. Cells were monitored in a humid tissue culture chamber at 37°C with CO2, and multipoint images were taken using a Nikon Elipse Ti fluorescent microscope with a Plan Fluor 10×/0.3 objective, ORCA-R2 camera, and MetaMorph software. Color composite time-lapse images were exported as movies using MetaMorph and viewed and evaluated using Fiji (ImageJ) software.
3
Expression of β2-Adrenergic Receptor in U2OS Cells
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U2OS cells (ATCC) were cultured in high-glucose DMEM (Dulbecco’s Modified Eagle medium, Thermo Fisher Scientific) supplemented with GlutaMAX-1 (Thermo Fisher Scientific) and 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) in a humidified 5% CO2 incubator at 37 °C. CAPAN-1 cells (ATCC) were cultured in McCoy’s 5a Medium Modified supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) in a humidified 5% CO2 atmosphere at 37 °C. Cells were split every 3–5 days or at confluence. Cells were seeded in glass bottom 12-well plates (MatTek Corporation). To express β2AR-YFP, U2OS cells were transiently transfected with plasmid pEYFP-β2AR (Addgene; Cat. No. 67146)48 (link) using Lipofectamine 2000 according to reverse transfection protocol adapted for plasmid DNA49 . We used 200,000 cells per well, 3 µg plasmid DNA and 3 µl of Lipofectamine 2000.
4
Time-Lapse Imaging of Cell Abscission
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For time-lapse phase-contrast imaging (abscission times), 30,000 cells were plated on glass-bottom 12-well plates (MatTek) 2 days before recording, incubated in an open chamber (Life Imaging) equilibrated in 5% CO2, and maintained at 37°C. In the case of high and low confluency conditions (Fig. 5E ), 100,000 and 10,000 trypsinized cells, respectively, were replated on glass-bottom 12-well plates for 3 hours. The culture medium was then changed, and SiR-tubulin was added for 1 hour before recording. Time-lapse sequences were recorded every 10 min for 48 hours using an inverted Nikon Eclipse Ti-E microscope with a 20× 0.45 NA Plan Fluor ELWD controlled by MetaMorph software (Universal Imaging).
For time-lapse fluorescent imaging, images were acquired using an inverted Nikon Eclipse Ti-E microscope equipped with a CSU-X1 spinning disk confocal scanning unit (Yokogawa) and with an electron-multiplying CCD (EMCCD) camera (Evolve 512 Delta, Photometrics) or a Prime 95S sCMOS camera (Teledyne Photometrics). Images were acquired with CSU 100× or CSU 60× 1.4 NA PL-APO VC objectives and MetaMorph software (MDS). For fig. S3B, images were acquired at 1-min intervals with Z-stacks (0.7 μm between Z-planes) for 3 hours. Cavin1 dots were manually tracked with ImageJ and analyzed from the Z-planes with the highest fluorescence intensities.
For time-lapse fluorescent imaging, images were acquired using an inverted Nikon Eclipse Ti-E microscope equipped with a CSU-X1 spinning disk confocal scanning unit (Yokogawa) and with an electron-multiplying CCD (EMCCD) camera (Evolve 512 Delta, Photometrics) or a Prime 95S sCMOS camera (Teledyne Photometrics). Images were acquired with CSU 100× or CSU 60× 1.4 NA PL-APO VC objectives and MetaMorph software (MDS). For fig. S3B, images were acquired at 1-min intervals with Z-stacks (0.7 μm between Z-planes) for 3 hours. Cavin1 dots were manually tracked with ImageJ and analyzed from the Z-planes with the highest fluorescence intensities.
5
DENV Infection Induces cGAS Localization Dynamics
6
Live-cell imaging of infected cell monolayers
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Light microscopy of infected cell monolayers was carried out as described53 (link), except for a few modifications. Glass bottom 12-well plates were obtained from MatTek Corporation and treated with 150 μg/ml poly-L-lysine. Monolayers were seeded and infected with bacteria in 200 μl of DMEM + FBS at an MOI of 10. After 1 h the medium containing bacteria was removed, the monolayers were washed two times with 1XPBS, and DMEM containing 750 μg/ml amikacin and 750 μg/ml kanamycin was added for the remainder of the experiment. Live-cell imaging was taken on an Olympus microscope equipped with the Weather Station incubation system at 37 °C with 5% CO2. Images were captured at 1 frame/5 min for 24 h. Images were compiled into videos using ImageJ.
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