Nostoc cultures were grown on agar plates and resuspended in BG11 medium. OD750 was adjusted to ~0.8 and 10 ml of the cultures were transferred into a petri dish. Cultures were treated with two Sankyo Denki Germicidal 68 T5 UV-C lamps for 5 mins in a repurposed Herolab UV DNA Crosslinker CL-1. Ten μl of Nostoc cultures were spotted on a BG11 agar plate and left until the liquid was fully absorbed. An area was cut out and mounted on a cell culture imaging dish. Time-lapse movies were recorded using a 40x objective on a Leica Thunder Imager 3D Cell Culture equipped with a Leica DFC9000 GTC CMOS camera (2048 × 2048 pixels, pixel size 6.5 μm). Images were recorded every 60 s over a time course of 5 h. To analyze the survival rate after UV-treatment on culture level, 3 ml of Nostoc cultures were treated 1 min with UV light and a dilution series was spotted on a pre-warmed BG11 plate. The plate was incubated in the dark for 24 h at 28 °C. Afterwards, incubation was continued for 10 days with continuous light.
Thunder imager 3d cell culture
Manufactured by Leica
The THUNDER Imager 3D Cell Culture is a high-performance imaging system designed for advanced 3D cell culture applications. It provides efficient and accurate imaging capabilities for a wide range of 3D cell culture models, including spheroids, organoids, and hydrogels.
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Lab products found in correlation
Dfc9000 gtc cmos camera, by Leica (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
L3224, by Thermo Fisher Scientific (3 mentions)
Cy2 conjugated goat anti mouse igg, by Dianova (2 mentions)
Las x premium imaging software, by Leica (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Fm5 95 dye, by Thermo Fisher Scientific (1 mentions)
Bx41 light microscope, by Olympus (1 mentions)
8 protocols using thunder imager 3d cell culture
1
Time-lapse Imaging of UV-Treated Nostoc
2
Immunofluorescence Staining of VeroE6 Cells
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VeroE6 cells were seeded and treated as described for the icELISA. After incubation with the primary antibody solution, 50 µL of secondary antibody solution (Goat IgG anti-Mouse IgG [H+L]-Alexa Fluor 488, MinX none 1:2,000 [RRID: AB_2338840], Phalloidin CF647 1:100 [Biotium] in PBS + 1% FBS) were added to each well, and the plate was incubated for 2 h at room temperature. Thereafter, the secondary antibody solution was aspirated, and the cells were counterstained for 20 min at room temperature with 50 µL of DAPI solution (0.1 µg/mL DAPI [Sigma-Aldrich] in PBS). Subsequently, the plate was washed thrice with PBS and microscopically analyzed using Leica THUNDER Imager 3D Cell Culture.
3
Confocal Imaging of Cell Cultures
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All in vivo images (except for Fig. 6 ; see Protein localization in tissue above) were obtained on a Zeiss LSM800 confocal microscope with a 40× Plan-Neofluar 1.3NA objective or 63× Plan-Apo 1.4NA objective. All images of Sf9 cells were obtained using a Leica THUNDER Imager 3D Cell Culture with Leica N Plan 5×/0.12 PH0 objective.
4
Visualizing SARS-CoV-2 Infection in Cells
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Cells were infected with SARS-CoV-2 and fixed after 20 or 30 h of infection using 4% (w/v) paraformaldehyde/PBS for > 2 h before they were discharged from the BSL-3 laboratory. Cells were permeabilized with 1% (v/v) Triton-X-100/PBS and blocked with 3% (v/v) FCS/PBS. SARS-CoV-2 infection was visualized by use of α-S mAb (kindly provided by Peter Miethe, fzmb, Bad Langensalza, Germany) and Cy2-conjugated goat anti-mouse IgG (Dianova). Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI; Sigma). Fluorescence was visualized using a THUNDER Imager 3D Cell Culture (Leica). Image analysis and processing were performed with LAS X Premium imaging software (Leica).
5
Organoid Viability Quantification
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To characterize viability, organoids were submerged in a solution consisting of DPBS supplemented with 2 µM calcein AM and 4 µM ethidium homodimer for 20 min at 37 °C (Thermo Fisher Scientific L3224). The samples were washed with DPBS and imaged with an epifluorescent microscope (Leica Microsystems, THUNDER Imager 3D Cell Culture) and confocal microscope (Leica SPE).
6
Bacterial Cultures on Agar Pads
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For sample preparation, 1.5% agar pads were freshly poured into a 35-mm-high glass-bottom µ-dish (ibidi). After 30 min, the agar pad was inverted and five 1.5 µl drops of respective bacterial culture were equally spotted onto the pad. The bacterial cultures were adjusted to an OD600 of 0.05 for fLM experiments and to an OD600 of 0.01 for timelapse imaging. After drying, the agar pad was mounted on a 35-mm-high glass-bottom µ-dish (ibidi) supplemented with a wet Kim wipe and closed with parafilm and vacuum grease.
Images were recorded using a ×100 phase-contrast objective on a Leica Thunder Imager 3D Cell Culture equipped with a Leica DFC9000 GTC CMOS camera (2,048 × 2,048 pixels, pixel size 6.5 mm) at a stage temperature of 25 °C. The Leica Application Suite X (LAS X) software platform was used for acquisition and the resulting images were analysed using Fiji65 (link), GraphPad Prism and Microsoft Excel. Different cell types were quantified using the cell counter function in Fiji. For timelapse imaging of Y. entomophaga chi2-sfGFP, single images were recorded every 10, 15 or 20 min over several hours using a highspeed software autofocus with a local range of 25 µm using the brightfield channel.
Images were recorded using a ×100 phase-contrast objective on a Leica Thunder Imager 3D Cell Culture equipped with a Leica DFC9000 GTC CMOS camera (2,048 × 2,048 pixels, pixel size 6.5 mm) at a stage temperature of 25 °C. The Leica Application Suite X (LAS X) software platform was used for acquisition and the resulting images were analysed using Fiji65 (link), GraphPad Prism and Microsoft Excel. Different cell types were quantified using the cell counter function in Fiji. For timelapse imaging of Y. entomophaga chi2-sfGFP, single images were recorded every 10, 15 or 20 min over several hours using a highspeed software autofocus with a local range of 25 µm using the brightfield channel.
7
Cell Diameter Measurement by Microscopy
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Phase-contrast microscopy was performed using Olympus BX41 light microscope. Fluorescent microscopy was performed using Leica Thunder Imager 3D Cell Culture equipped with a Leica DFC9000 GTC CMOS camera. DNA of the cells was strained by NucBlue™ LiveReadyProbes™ Reagent (Life Technologies #R37605) according to the manufacturer's recommendations, prior to microscope analysis. To stain the cytoplasmic membrane, FM 5-95 dye (Invitrogen #T23360) was added to cell suspension to a final concentration 5 μg ml À1 and incubated for 1 min before analysis. Cell diameter was measured manually by taking a random diameter using Fiji software (Schindelin et al., 2012) .
8
Visualizing SARS-CoV-2 Infection in Cells
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Cells were infected with SARS-CoV-2 and fixed after 20 or 30 h of infection using 4% (w/v) paraformaldehyde/PBS for >2 h before they were discharged from the BSL-3 laboratory.
Cells were permeabilized with 1% (v/v) Triton-X-100/PBS and blocked with 3% (v/v) FCS/PBS. SARS-CoV-2 infection was visualized by use of α-S mAb (kindly provided by Peter Miethe, fzmb, Bad Langensalza, Germany) and Cy2-conjugated goat anti-mouse IgG (Dianova). Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI; Sigma). Fluorescence was visualized using a THUNDER Imager 3D Cell Culture (Leica). Image analysis and processing were performed with LAS X Premium imaging software (Leica).
Cells were permeabilized with 1% (v/v) Triton-X-100/PBS and blocked with 3% (v/v) FCS/PBS. SARS-CoV-2 infection was visualized by use of α-S mAb (kindly provided by Peter Miethe, fzmb, Bad Langensalza, Germany) and Cy2-conjugated goat anti-mouse IgG (Dianova). Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI; Sigma). Fluorescence was visualized using a THUNDER Imager 3D Cell Culture (Leica). Image analysis and processing were performed with LAS X Premium imaging software (Leica).
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