Cells were imaged in 96-well glass-bottom microplates (Greiner Bio-One) containing indicated media with an inverted microscope (Ti-E; Nikon) using a Plan Apochromat IR 60× 1.27 NA objective (Nikon) and Spectra X LED light source (Lumencor) at room temperature. 3D light microscopy data were collected using the triggered Z-Piezo system (Nikon) and orca flash 4.0 camera (Hamamatsu). 3D data were processed using NIS-elements AR (Nikon), Huygens Professional 16.10 (Scientific Volume Imaging), ImageJ v.2.0.0., and Photoshop (Adobe Systems) software.
Spectrax led light source
Manufactured by Lumencor
Sourced in Ireland
The SpectraX LED light source is a compact, high-performance illumination device designed for laboratory and scientific applications. It employs multiple LED light engines to generate a wide range of illumination wavelengths. The SpectraX provides stable, uniform, and adjustable light output, making it suitable for various microscopy, spectroscopy, and other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Z piezo system, by Nikon (3 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (3 mentions)
Plan apochromat ir 60 1.27 na objective, by Nikon (2 mentions)
96 well glass bottom microplates, by Greiner (2 mentions)
Nis elements ar, by Nikon (2 mentions)
Orca flash4.0 lt plus scmos camera, by Hamamatsu Photonics (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Huygens professional 16, by Scientific Volume Imaging (1 mentions)
Element software, by Nikon (1 mentions)
Ti2 inverted microscope, by Nikon (1 mentions)
1.5 glass, by Cellvis (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Mycoplasma detection kit, by Lonza (1 mentions)
Orca flash 4.0 scmos camera, by Hamamatsu Photonics (1 mentions)
Fugene, by Roche (1 mentions)
Cy3 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Zyla 4.2 plus scmos camera, by Oxford Instruments (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Hydromount, by National Diagnostics (1 mentions)
Alexa fluor 488 cy3, by Jackson ImmunoResearch (1 mentions)
10 protocols using spectrax led light source
1
3D Live-Cell Microscopy Imaging
2
Live-cell imaging of mitotic response
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RPE1 cells stably expressing Histone H2B-RFP or MDA-MB-231 cells stably expressing Histone H2B-mCherry were plated on 4-chaamber 35 mm glass bottom dish at least one day prior to do imaging (#1.5 glass, Cellvis). Cells were treated with DMSO (control), PTX (10 nM), or ERI (10nM) 1 hour prior to imaging. High-temporal live-cell imaging was performed using a Nikon Ti2 inverted microscope equipped with a Hamamatsu Fusion camera, spectra-X LED light source (Lumencor), Shiraito PureBox (TokaiHit) and a Plan Apo 20x objective (NA = 0.75) controlled by Nikon Element software and Metamorph (Molecular Devices). Cells were recorded at 37°C with 5% CO2 in a stage-top incubator using the feedback control to maintain the growing media’s temperature (Tokai Hit, STX model). Image analysis was performed using Nikon Element software. Mitotic stages were determined by nuclear staining. The mitotic duration was measured from nuclear envelope breakdown (NEBD) to anaphase onset. Incidences of multi-nuclei, mitotic slippages and unaligned chromosome were analyzed. The experiments were independently repeated 2–3 times for mitotic duration measurements (total of n = 100), and P-values between variants were calculated by One-Way Anova and two-tailed t-test. P-values < 0.05 were considered significant.
3
High-resolution 3D microscopy of cells
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Cells were viewed in 96-well microplates with a glass bottom (Greiner bio-one) containing indicated growth or starvation media at RT with an inverted microscope (Ti-E; Nikon) using a Plan Apochromat IR 60× 1.27 NA objective (Nikon), Spectra X LED light source (Lumencor), and acquisition software NIS elements AR (Nikon). 3D light microscopy data were collected using the triggered Z-Piezo system (Nikon) and orca flash 4.0 camera (Hamamatsu). 3D data were processed using NIS-elements AR; deconvolution of imaging data was performed with 3D deconvolution 3D-Fast in NIS-elements AR (Figs. 2 E , 3 C , 4 A , and 5 A ) or Huygens Professional 15.10 (Scientific Volume Imaging; Figs. 1, B and C ; Fig. 2 C ; Fig. 4 D ; and Fig. S1, A, C, and G). Photoshop (Adobe) software was used to make linear adjustments in brightness.
4
Assessing C. difficile Toxin Activity
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C. difficile toxin activity was assessed by Vero cell rounding. Vero (ATCC CCL-81) cells were obtained from ATCC and grown in Dulbecco’s Modified Eagle Medium (ThermoFisher) supplemented with 10% fetal bovine serum (FBS) at 37°C, 5% CO2. Vero cells were routinely tested for Mycoplasma using the Mycoplasma Detection Kit (Lonza, cat# LT07-518). For cell rounding assays, Vero cells were grown to confluency on 96-well plates. Once confluent, the medium was changed to DMEM without FBS and C. difficile CDMM supernatant was used to treat the Vero cells (final concentration of 50% C. difficile supernatant in DMEM). Cells were imaged after 4 hr incubation on a Nikon TiE inverted widefield epifluorescence microscope (Nikon) using a SPECTRAX LED light source (Lumencor). Fluorescence and transmitted light images were recorded using an ORCA-Flash 4.0 sCMOS camera (Hamamatsu), and Nikon Elements Advanced Research v4.5 software was used for data acquisition. For analysis, FIJI (Formerly Image J; National Institutes of Health) was used to define cell shapes and cell diameter was recorded in three regions/well, n = 4 wells; repeated two independent times. Cell rounding was defined as a > 50% reduction in cell diameter.
5
Lentiviral Transduction of TP53 and MYC
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Creation of this model is described in detail in the main body of the manuscript. The cells are grown in EGF/FGF media made with a 50:50 mixture of F12:DMEM, but is otherwise identical to that described above. The R248W-TP53 plasmid (Addgene plasmid 16437) and MYC plasmid (Addgene plasmid 17758) were subcloned into pWPI (Addgene 12254) [29] (link). Lentivirus was produced by transfecting 293T cells with VSV-G envelope plasmid, Δ8.9 gag/pol plasmid and the plasmid containing the gene of interest as described in [36] (link) using Fugene (Roche) per manufacturer’s instruction. The supernatant was collected at 48 and 72 h. The collected supernatant was filtered with a 0.45 micron filter and concentrated overnight at 4’C using 5% PEG 8000 and 150 mM NaCl then centrifuged for 30 minutes at 2000xg. Concentrated virus was stored at -80C in serum free DMEM until use. Visualization of the distribution of this model in situ was provided by fluorescence imaging macroscopy using a dual camera setup for GFP and white light (anatomical) imaging. The excised brain was illuminated by a SpectraX LED light source (Lumencor) with emission light captured by an F1.2 80 mm lens (Nikon), and filtered by a dichroic and low pass filter in an OptosplitII (Cairn) tube to dual Pixelfly CCDs (PCO). Data was acquired in Metamorph NX.
6
Retinal Immunocytochemistry and Imaging
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Mice were sacrificed, eyes enucleated and fixed overnight in 4% paraformaldehyde in PBS before the retinas were removed from the eyecups and immunocytochemistry performed as described previously described [58 (link)]. Whole retinas were incubated with anti-BRN3A primary antibody (Synaptic Systems 411003, Goettingen, Germany; 1/200 dilution) [59 (link),60 (link)] for 3 days at 4 °C, washed in PBS and then incubated with Cy3 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories; 1/400) for 2 days at 4 °C. Wholemount and cell images were taken using an Olympus IX83 inverted motorised microscope (Mason Technology, Dublin, Ireland) equipped with a SpectraX LED light source (Lumencor, Mason Technology, Dublin, Ireland) and an Orca-Flash4.0 LT PLUS/sCMOS camera (Hamamatsu, Tsukuba City, Japan), as previously described [47 (link)]. Samples were imaged using a 4x (wholemount) and 40× (cells) objective utilising enhanced focal imaging (EFI) with typically 5–8 Z-slices. Lateral frames for wholemounts were stitched together and analysed in Olympus CellSens software (v1.9; Waltham, MA, USA). Cell counting was performed utilizing 2D deconvolution, manual threshold and object size filter, with the same settings/operations applied to all images.
7
High-Resolution Microscopy of Cellular Dynamics
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Cells were transferred to 96-well glass-bottom microplates (Greiner Bio-One) containing indicated media. For fixed timepoint analyses, cells were imaged at room temperature with an inverted microscope (Nikon Ti-E) using a Plan Apochromat IR 60× 1.27 numerical aperture objective (Nikon) and Spectra X LED light source (Lumencor). Three-dimensional light microscopy data were collected using the triggered Z-Piezo system (Nikon) and orca flash 4.0 camera (Hamamatsu). High-speed confocal imaging was performed with a Dragonfly 500 series spinning disk microscope (Andor, Oxford Instruments) equipped with a Zyla 4.2 Plus sCMos camera (Andor) using a Lamba CFI-Plan Apochromat 60× 1.4 numerical aperture oil immersion objective (Nikon). Three-dimensional data were processed using Fusion software (Andor), and Fiji ImageJ Version 2.1.0. Deconvolution was performed using Huygens Professional 16.10 where indicated. Data are shown as single sections if not stated otherwise.
8
Rotavirus Infection in Cell Monolayers
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Cell monolayers were grown in Ibidi 8-well chamber slides. On the day of imaging, media were replaced with FluoroBrite-Plus media [FluoroBrite DMEM supplemented with 1× nonessential amino acids, 1× Glutamax, 1× sodium pyruvate, and 15 µM HEPES (Invitrogen)]. Rotavirus was diluted in FluoroBrite-Plus, and cells were mock- (FluoroBrite-Plus alone) or rotavirus-infected at MOI 0.01. The inoculum was removed after 1 h, fresh FluoroBrite-Plus was added, and the slide was mounted into the microscope environmental chamber (37°C in 5% CO2) to equilibrate. Imaging was performed using a Nikon TiE inverted microscope using a SPECTRAX LED light source (Lumencor) and either a 20× Plan Fluor (NA 0.45) or a 20× Plan Apo (NA 0.75), objective as previously described (21 (link)). Nikon Elements Advanced Research v4.5 software was used for data acquisition and image analysis. Fluorescence intensity values were exported to Microsoft Excel and GraphPad Prism for data analyses.
9
Retinal Ganglion Cell Immunostaining
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Mice were sacrificed 3 days after OKR assessment and eyes were enucleated and fixed in 4% paraformaldehyde in PBS overnight. Eyes were washed in PBS, then the retinas were removed from the eyecups and immediately processed for immunocytochemistry. Immunocytochemistry was performed as described previously (Palfi et al., 2016 (link)). Whole retinas were incubated with primary antibodies for RBPMS (ABN1376, Millipore, 1:200; Rodriguez et al., 2014 (link)) overnight for 3 days at 4°C. Retinas were then washed in PBS and incubated with secondary antibodies conjugated with Alexa-Fluor-488, Cy3 (Jackson ImmunoResearch Laboratories; 1:400) for 2 days and nuclei counterstained with DAPI. Samples were covered using Hydromount (National Diagnostics). Fluorescent microscopy was carried out utilizing an Olympus IX83 inverted motorized microscope (cellSens v1.9 software) equipped with a SpectraX LED light source (Lumencor) and an Orca-Flash4.0 LT PLUS/sCMOS camera (Hamamatsu). Samples were imaged using a 10x plan fluorite objective utilizing enhanced focal imaging (EFI) with typically 5–8 Z-slices. Lateral frames were stitched together and analyzed in cellSens. Automated cell staining area was calculated utilizing 2D deconvolution, manual threshold and object size filter in cellSense; the same settings/operations were applied to all images.
10
BCECF Fluorescence Imaging for pH Measurement
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Cells were grown in SDC as noted above and treated with 5 µM BCECF (Thermo Fisher). BCECF fluorescence was recorded on a Leica DMi8 equipped with a pco.edge 4.2 camera (pco), a SpectraX LED light source (Lumencor) and a ×60 objective. Both pH-sensitive and insensitive BCECF fluorescence was obtained using a 540/21 emission filter and a FF444/520/590-Di01 dichroic mirror (Semrock). pH-insensitive fluorescence was obtained illuminating the sample with a 427/10 bandpass filter and pH-sensitive fluorescence used a 540/21 bandpass filter. For both fluorescence signals, incident light was attenuated to 30% power and an exposure times of 45 ms was used.
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