Viral infections were imaged with an Amersham Typhoon 5 scanner (Cytiva) with the default Cy2 (excitation, 488 nm; emission, 520 nm) setting. Images of cells infected with SINV BFP-to-GFP edited virus were captured with Nikon Ti-Eclipse inverted microscope (Nikon Instruments) equipped with a SpectraX light-emitting diode (LED) excitation module (Lumencor) and emission filter wheels (Prior Scientific). Fluorescence imaging used excitation/emission filters and dichroic mirrors for GFP and 4′,6-diamidino-2-phenylindole (DAPI) (Chroma Technology Corp.). Images were acquired with Plan Fluor 20Ph objective and an iXon 896 electron-multiplying charge-coupled device (EM-CCD) camera (Andor Technology Ltd.) in NIS-Elements software. Nucleic acid gels were stained with SYBR Gold (Thermo Fisher Scientific) and imaged using the Cy2 setting with an Amersham Typhoon 5 scanner (Cytiva). Protein gels were stained with Coomassie stain (in-house), washed, and imaged with infrared-short default setting with an Amersham Typhoon 5 scanner (Cytiva).
Amersham typhoon 5 scanner
Manufactured by Cytiva
Sourced in Germany
The Amersham Typhoon 5 scanner is a versatile laboratory imaging system designed for a range of applications, including protein and nucleic acid detection, phosphor imaging, and autoradiography. It utilizes laser-based detection technology to capture high-quality images of various samples.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant tl software, by Cytiva (3 mentions)
Emission filter wheels, by Prior Scientific (2 mentions)
Plan fluor 20ph objective, by Oxford Instruments (2 mentions)
Sybr gold, by Thermo Fisher Scientific (2 mentions)
Eclipse ti inverted microscope, by Nikon (2 mentions)
Emccd camera, by Oxford Instruments (1 mentions)
Spectra x, by Lumencor (1 mentions)
Hinfi, by New England Biolabs (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
Seakem le agarose gel, by Lonza (1 mentions)
7 protocols using amersham typhoon 5 scanner
1
Imaging Viral Infections and Biomolecules
2
Virus Imaging and Gel Analysis
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Viral infections were imaged with Amersham Typhoon 5 scanner (Cytiva) with default Cy2 (excitation 488 nm, emission 520 nm) setting. Images of cells infected with SINV BFP-to-GFP edited virus were captured with Nikon Ti-Eclipse inverted microscope (Nikon Instruments) equipped with a SpectraX LED excitation module (Lumencor) and emission filter wheels (Prior Scientific). Fluorescence imaging used excitation/emission filters and dichroic mirrors for GFP and DAPI (Chroma Technology Corp.). Images were acquired with Plan Fluor 20Ph objective and an iXon 896 EM-CCD camera (Andor Technology Ltd.) in NIS-Elements software. Nucleic acid gels were stained with SYBR Gold (ThermoFisher Scientific) and imaged using Cy2 setting with Amersham Typhoon 5 scanner (Cytiva). Protein gels were stained with Coomassie stain (in-house), washed and imaged with IR-short default setting with Amersham Typhoon 5 scanner (Cytiva).
3
Clonogenic Potential of LNCaP Cells Exposed to 225Ac-Radioconjugates
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Colony formation of LNCaP cells upon exposure to increasing activity concentrations of both 225Ac-radioconjugates was analyzed as previously reported 25 (link) with slight modifications. Briefly, 6000 LNCaP cells were seeded in 6-well microplates (Greiner Bio-One GmbH, Frickenhausen, Germany) and cultivated overnight to allow cell adhesion. The cell culture medium was replaced by serum-free RPMI-1640 medium (2 mL/well) and the microplates were further incubated at 37 °C. After 1 h, five different activity concentrations (0.05, 0.5, 1, 5, and 50 kBq/mL) of the 225Ac-radioconjugates were added in triplicate. After 1 or 4 h of incubation at 37 °C, the supernatants were replaced by fresh RPMI-1640 medium with 10% FCS (2 mL/well) and plates were incubated at 37 °C for 8 d. Finally, the cell culture medium was discarded and colonies were stained with 0.5% crystal violet in 50% methanol (1 mL/well) for 30 min, after which the plates were rinsed three times with deionized water and subsequently air-dried. The plates were scanned with an Amersham Typhoon 5 Scanner (Cytiva Europe GmbH, Freiburg, Germany) and the colonies were counted using the Image-Quant TL software (Version 8.1, Cytiva Europe GmbH, Freiburg, Germany).
4
Plaque Isolation and Amplification Protocol
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The cells electroporated with edited viral RNA were serially diluted, and 10,000 cells were seeded on a monolayer of BHK-21 cells in a 100-mm petri dish. The cells were incubated (37°C, 5% CO2) for 1 hour. Next, the medium was removed, and the medium supplemented with 0.3% agarose was overlaid. Petri dishes were imaged 16 to 18 hours after infection using an Amersham Typhoon 5 scanner (Cytiva). The individual plaques were picked using sterile tips under a light microscope and inoculated in naive BHK-21 cells for propagation.
5
Virus Propagation via Plaque Assay
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The cells electroporated with edited viral RNA were serially diluted, and 10,000 cells were seeded on a monolayer of BHK-21 cells in a 100 mm Petri dish. The cells were incubated (37°C, 5% CO2) for one hour. Next, the media was removed, and media supplemented with 0.3% agarose was overlayed. Petri dishes were imaged 16–18 hours post-infection using Amersham Typhoon 5 scanner (Cytiva). The individual plaques were picked using sterile tips under a light microscope and inoculated in naive BHK-21 cells for propagation.
6
Clonogenic Assay of Radiolabeled PSMA Ligands
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LNCaP cells were plated in 6-well cell culture microplates (Greiner Bio-One GmbH, Frickenhausen, Germany) at a density of 6 × 103 cells/2 mL/well. After 24 h, four different activity concentrations (0.05, 0.5, 5 and 50 kBq/mL) of the 225Ac-labeled ligands [225Ac]Ac-mcp-M-PSMA and [225Ac]Ac-mcp-D-PSMA were added in triplicate. After 1 or 4 h of incubation, the cell culture media was removed and fresh media was added to each well. Plates were then incubated at 37 °C for 8 days and re-fed every 3 to 4 days. Finally, the cell media was removed and colonies were stained with 1 mL of 0.5% crystal violet in 50% methanol for 30 min, after which the plates were rinsed three times with deionized water and subsequently air-dried. The plates were scanned with an Amersham Typhoon 5 Scanner (Cytiva Europe GmbH, Freiburg, Germany) and the colonies were counted using the Image-Quant TL software (Version 8.1, Cytiva Europe GmbH, Freiburg, Germany).
7
Telomere Length Analysis of iPSCs
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Genomic DNA (2.2 µg) from iPSCs was digested with Hinf I (New England Biolabs, Cat. No: #R0155S) and Rsa I (New England Biolabs, Cat. No: #R0167L) restriction enzyme in 10X CutSmart® Buffer (NEB, Cat. No: #B7204S) at 37 °C overnight. The digested gDNA fragments were separated on a 1% SeaKem® LE agarose gel (Lonza, Cat. No: 50002) by electrophoresis at 120 V for 12 h, followed by capillary transfer to a HybondTM-N+ nylon transfer membrane (GE Healthcare, Cat. No: RPN303B) in 10X SSC for 14.5 h. DNA was subsequently crosslinked to the membrane twice at 120 mJ in a UV Stratalinker 1800 (Stratagene, 254 nm, 120 mJ). The blot was prehybridized in Church buffer at 65 °C for an hour and then hybridized with 32P-α-dCTP-labelled (TTAGGG)3 overnight. The blot was exposed to a phosphor image screen (Fujifilm) at room temperature overnight. Phosphor images were obtained with an Amersham Typhoon 5 scanner (Cytiva). The telomere length images were quantified and analysed by ImageQuantTL software (Cytiva). All blots derive from the same experiment and were processed in parallel.
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