SH-SY5Y cells were seeded on chambered coverglass (Thermo Fisher Scientific, NY, Cat 155380) using DMEM complete with tet. The following day the medium was replaced with OGD medium and the cells were subjected to OGD for 10 minutes. Immunostaining was carried out using anti-p65 antibody (Santa Cruz Biotechnology, Cat sc-372) as a primary antibody and Alexa Fluor®488 (Molecular Probes) (in green) coupled goat anti-rabbit IgG as a second antibody. Nuclear staining (in blue) was performed using a mounting medium with DAPI (Vector Laboratories Inc, Burlingame, CA). Images were captured with an oil immersion 40x objective on a Zeiss LSM510 microscope using LSM Image Software.
Chambered coverglass
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Chambered coverglass is a laboratory equipment used for various cell-based applications. It consists of a glass slide with a pre-formed chamber, providing a controlled environment for culturing and observing cells under a microscope. The chambered coverglass allows for easy media exchange and sample handling, making it a versatile tool for researchers in the field of cell biology.
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Lab products found in correlation
Lsm 710, by Zeiss (6 mentions)
Lsm 780, by Zeiss (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Incubation chamber, by Pecon (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Axiocam 506, by Zeiss (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Famotidine, by Merck Group (2 mentions)
Omeprazole, by Merck Group (2 mentions)
Histamine, by Merck Group (2 mentions)
Snarf 5f, by Thermo Fisher Scientific (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Sp5 confocal microscope, by Leica camera (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
58 protocols using chambered coverglass
1
OGD-induced p65 nuclear translocation
2
Curcumol Effects on HSC Organelles
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HSCs were inoculated into 4-well Chambered Coverglass (Thermo Scientific, Waltham, MA, USA) with a cell density of 2 × 104 cells/mL (14,000 cells/well), and then TEM was used to observe the changes in organelle morphology of HSCs after curcumol treatment (Zhang et al., 2018b (link)).
3
HOTAIRM1 Regulation of DNA Damage Response
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U2OS cells were seeded in a chambered coverglass (Thermo Scientific) and transiently transfected with pcDNA-HOTAIRM1-6× MS2 (full-length, Δ5′, ΔE2 or Δ3′) and pcDNA-MCP-GFP or GapmeRs. Laser microirradiation was performed 48 h post-transfection using a laser-scanning confocal microscope (LSM 780, Carl Zeiss) and a 405 nm laser diode. After laser microirradiation, cells were fixed with 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 in PBS. Immunofluorescence was performed by sequential incubation with primary and secondary antibodies. Nuclei were counterstained in Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Samples were visualized using a laser-scanning confocal microscope (LSM 780, Carl Zeiss) coupled with an image analysis system.
For live cell imaging, U2OS cells that stably expressed GFP-fused Ku70, Ku80 or MDC1 (33 (link)) were transiently transfected with pLKO.1-shHOTAIRM1. Laser microirradiation and time lapse imaging were carried out in a SP5 X inverted confocal microscope (Leica Microsystems) using laser diodes at 405 nm and 488 nm, respectively.
For live cell imaging, U2OS cells that stably expressed GFP-fused Ku70, Ku80 or MDC1 (33 (link)) were transiently transfected with pLKO.1-shHOTAIRM1. Laser microirradiation and time lapse imaging were carried out in a SP5 X inverted confocal microscope (Leica Microsystems) using laser diodes at 405 nm and 488 nm, respectively.
4
Live-cell Imaging and Lysotracker Assay for HCC
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For live-cell imaging, HCC cell lines and primary human hepatocytes were seeded (1.5 × 104 cells per well) in a chambered coverglass (Thermo Scientific) and treated as indicated before. After 24 h cells were washed twice in cold DPBS and stained with LysoTracker DNS-99 (Thermo Fisher). Nuclei were counterstained with DAPI (1:5000). Verteporfin auto-fluorescence was observed in the far-red spectrum (Cy5 channel). Fluorescence images of live-cells were obtained using an automated inverted microscope (Leica DMSI4000 B). For the Lysotracker fluorescence assay, HCC cells were seeded (1.5 × 104 cells per well) on a 96-well assay plate and treated as described. After 24 h, medium was aspirated and discarded from all the wells followed by Lysotracker incubation as previously reported30 (link). LysoTracker fluorescence intensity was then measured by spectrophotometry (Ex = 577 nm, Em = 590 nm) on a fluorescence plate reader (Tecan Infinite® 200).
5
Visualizing SARM1 activity in HEK293 cells
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HEK293 cells, overexpressing wildtype or the enzymatically dead form (E642A) of SARM1 or HEK293T Knocking out NMNAT1 were constructed as before (Zhao et al., 2019 (link)). Cells, grown on 0.05 mg/mL poly-L-lysine coated Chambered coverglass (ThermoFisher, #155411) overnight, were treated with 50 μM PC6 in the presence or absence of 100 μM CZ-48 for 8 hr (for SARM1-OE cells) and 200 μM CZ-48 for 48 hr (for wildtype HEK293T cells), respectively. To demonstrate the edges of the cells, they were stained with 50 μg/mL Concanavalin A, Alexa Fluor 647 Conjugate (ThermoFisher) at 4°C for 10 min before imaging. The fluorescence signals (Ex/Em: 405/525 nm for PAD6; Ex/Em: 561/590 for ConA) were captured under a confocal microscope (Nikon A1).
6
Visualizing Seedling Cell Wall Composition
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Vertically grown 5‐day‐old seedlings were incubated in methanol for 3 days at room temperature. The cleared seedlings were transferred to a freshly prepared solution of Fluorol Yellow 088 (0.01%, in methanol) and incubated for 1 h. The stained seedlings were rinsed shortly in methanol and transferred to a freshly prepared solution of aniline blue (0.5%, in methanol) for counterstaining. Finally, the seedlings were washed for 2–3 min in water and transferred to a chambered coverglass (Thermo Scientific), covered with a piece of 1% half‐strength MS agar, and imaged using a Zeiss LSM 880 confocal microscope as described above.
7
Immunofluorescence Assay of Huh7 Cells
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Cell immunofluorescence assay was performed as previously described (Khakpooret al., 2009; Gao et al., 2015 (link)). Briefly, treated Huh7 cells were cultured on a chambered coverglass (Thermo Fisher Scientific) and then fixed with 4% paraformaldehyde for 15 min at RT. The samples were permeabilized with 0.1% Triton X-100 for 5 min at RT and then blocked with 3% BSA (in PBS) for 2 h. Afterward, they were incubated with the indicated primary antibodies (2 h at RT) and fluorophore-conjugated secondary antibodies (1 h at RT) in 3% BSA and then stained with DAPI. The samples were observed using a Zeiss LSM 800 confocal fluorescence microscope. Images were adjusted for brightness and contrast and collocated into figures by using Photoshop CS8.
8
Maintaining HeLa Cells in DMEM
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HeLa CCL-2 cells (ATCC) were maintained in Dulbelcco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin in a humidified atmosphere at 37°C with 5% CO2. Cells were plated on chambered coverglass (0.2 ml medium/chamber) (Thermo Fisher Scientific) and allowed to reach 60% confluency in 1–2 days.
9
Quantification of DNA Damage Foci in Irradiated Cells
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The cultured cells on Chambered Coverglass (Thermo Scientific) were irradiated with 1 Gy of X-ray using the Pantak HF 350 X-ray generator (Shimadzu). One hour after X-ray irradiation, the cultured cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 15 min. The cells were blocked with 10% goat serum for 1 h and reacted with the anti-phospho-histone H2A.X (Ser139) antibody clone JBW301 (Merck Millipore, 05-636, at 1/800 dilution) for 1 h and then with Alexa Fluor 488 anti-mouse IgG (Molecular Probes, A-11001, at 1/500 dilution) for 45 min. Nuclear DNA was counterstained with 4′,6-diamidino-2-phenylindole (Invitrogen). All reactions and procedures were essentially performed at RT. Fluorescent signals were observed by confocal microscopy (LSM710, Carl Zeiss). The depth-coded projections were captured as stacks of ten optical sections of z-series at 1-μm intervals and converted to binarized images by ImageJ version 1.47. The threshold value for image conversion was manually adjusted until a visual best fit between the original and converted images was observed (Supplementary Fig. 22 ). The numbers of γ-H2AX foci were counted using the ImageJ software79 (link).
10
Visualizing GFP-Y14 Dynamics in U2OS Cells
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U2OS cells were cultured in Dulbecco's modified Eagle's medium supplemented with penicillin-streptomycin and 10% fetal bovine serum (Gibco), and transiently transfected with the vector encoding GFP-Y14 (wild-type or mutant) using lipofectamine 2000 (Thermo Fisher Scientific). The transfectants were observed using a laser-scanning confocal microscope (LSM 780, Carl Zeiss) coupled with an image analysis system. For FRAP, transfected U2OS cells were seeded in Chambered Cover glass (Thermo Fisher Scientific) for 48 h. Photobleaching was performed using a laser-scanning confocal microscope (LSM 780, Carl Zeiss) with a 488-nm laser diode. Images were continuously taken during 100 cycles for a total time of 200 sec. FRAP analysis and fluorescence intensity measurement were done using ZEN Black Software (Carl Zeiss).
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