Whole cell lysates were prepared in radioimmunoprecipitation (RIPA) buffer with added protease inhibitors (Roche). Protein concentrations were determined using the Bio-Rad DC Protein Assay (Bio-Rad). 30-40 μg of protein lysates were separated on 10 % (lab-made) or 14 % (ThermoFisher) SDS PAGE gels and electroblotted to PVDF membranes. Data were captured and analyzed by Carestream Image Station 4000 R Pro with Carestream Molecular Imaging Software, version 5.0, (Carestream Health, Inc). The values from regions of interest (ROI) normalized to the loading control and the normalized value of MCF-7 cells was set to 1 for comparison of cell lines between separate experiments.
Molecular imaging software version 5
Manufactured by Carestream
Sourced in United States
Carestream Molecular Imaging software version 5.0 is a comprehensive software solution designed for the analysis and processing of molecular imaging data. The software provides core functionalities for image visualization, quantification, and data management. It supports the integration and analysis of various molecular imaging modalities.
Automatically generated - may contain errors
Lab products found in correlation
Xenolight rediject d luciferin substrate, by PerkinElmer (3 mentions)
Immuno blot pvdf membrane, by Bio-Rad (2 mentions)
Image station 4000r pro, by Carestream (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Chemiluminescence western blotting kit, by Roche (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Chemiluminescent western blotting kit, by Roche (1 mentions)
Female balb c mice, by Charles River Laboratories (1 mentions)
7 protocols using molecular imaging software version 5
1
Protein quantification and Western blot
2
Quantitative Analysis of Tumor Size
3
Western Blot Analysis of NSCLC Cells
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NSCLC cells were treated, collected, and lysed by using the RIPA buffer (Cell Signaling Technology) containing the protease inhibitor cocktail. Protein concentrations were determined by using the BCA assay kit (Thermo Scientific). 50 μg of proteins from each treatment condition were electrophoresed on 9 % SDS–Polyacrylamide gels, and then transferred onto the Immuno-Blot PVDF Membrane (Bio-Rad). The membranes were probed with polyclonal antibodies, followed by detection with a chemiluminescence Western blotting kit (Roche Diagnostics). The signals were detected by using a Carestream image station 4000MM Pro, and quantitation was performed by using the Carestream molecular imaging software version 5.0.5.30 (Carestream Health, Inc.). Antibodies used were purchased from Santa Cruz Biotechnology and Sigma-Aldrich.
4
TAP-Pulldown of Yeast Protein Complexes
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Yeast cells expressing various TAP-tagged proteins and full-length Gis1 or Gis1 deletion proteins were grown in synthetic complete media to an optical density (600 nm) of approximately 1.0–1.5. Cells were harvested and extracts were prepared as described previously [71 (link)]. For TAP-pulldown, protein extracts were incubated with IgG-Sepharose beads to capture the target proteins. After this binding reaction, the beads were washed to remove unbound proteins, as described [72 (link)]. All steps were carried out in the cold room at 4 °C. Input and protein complexes bound to the beads were detected by Western blotting, where 75 μg of proteins from each treatment condition were electrophoresed on 10% SDS–Polyacrylamide gels, then transferred onto an Immuno-Blot PVDF Membrane (Bio-Rad, Hercules, CA, USA). The membranes were probed with antibodies, followed by detection with a chemiluminescent Western blotting kit (Roche Diagnostics, Mannheim, Germany). The signals were detected with a Carestream image station 4000MM Pro and quantitation was performed with the Carestream molecular imaging software version 5.0.5.30 (Carestream Health, Inc., Rochester, NY, USA).
5
Bioluminescent Skin Tissue Imaging
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Human skin tissue was collected and excised as described
above. Explants were incubated at 37 °C with 5% CO2 in Petri dishes with 10 mL of cDMEM. Medium was replaced daily.
Explants were injected ID using a Micro-Fine Demi 0.3 mL syringe (Becton
Dickinson, UK) with 2 μg of fLuc saRNA complexed with pABOL
or PEI in a volume of 100 μL. After 3 days, skin explants were
inverted and the medium was replaced with 5 mL of cDMEM supplemented
with 100 μL of XenoLight RediJectd -luciferin substrate
(PerkinElmer, UK) and imaged on an IVIS FX Pro (Kodak Co., Rochester,
NY, USA) equipped with Molecular Imaging software version 5.0 (Carestream
Health, USA) for 60 min. Signal from each injection site was quantified
using Molecular Imaging software and expressed as relative light units
(p/s).
above. Explants were incubated at 37 °C with 5% CO2 in Petri dishes with 10 mL of cDMEM. Medium was replaced daily.
Explants were injected ID using a Micro-Fine Demi 0.3 mL syringe (Becton
Dickinson, UK) with 2 μg of fLuc saRNA complexed with pABOL
or PEI in a volume of 100 μL. After 3 days, skin explants were
inverted and the medium was replaced with 5 mL of cDMEM supplemented
with 100 μL of XenoLight RediJect
(PerkinElmer, UK) and imaged on an IVIS FX Pro (Kodak Co., Rochester,
NY, USA) equipped with Molecular Imaging software version 5.0 (Carestream
Health, USA) for 60 min. Signal from each injection site was quantified
using Molecular Imaging software and expressed as relative light units
(p/s).
6
In Vivo Luciferase Imaging in Mice
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All animals were handled in accordance with the UK Home Office Animals
Scientific Procedures Act 1986 and with an internal ethics board and
UK government approved project (P63FE629C) and personal license (IC37CBB8F).
Food and water were supplied ad libitum. Female BALB/c
mice (Charles River, UK) 6–8 weeks of age were placed into
groups (n = 5) and housed in a fully acclimatized
room. In vivo imaging was performed as previously
described.43 (link) Mice were injected either
intramuscularly in both hind legs or intradermally with 5 μg
of fLuc saRNA complexed with either pABOL or PEI in a total volume
of 50 μL. After 7 days, the mice were injected intraperitoneally
(IP) with 100 μL of XenoLight RediJectd -luciferin
substrate (PerkinElmer, UK) and allowed to rest for 10 min. Mice were
then anesthetized using isoflurane and imaged on an In Vivo Imaging System (IVIS) FX Pro (Kodak Co., Rochester, NY, USA) equipped
with Molecular Imaging software version 5.0 (Carestream Health, USA)
for 2 min. Signal from each injection site was quantified using Molecular
Imaging software and expressed as relative light units (p/s).
Scientific Procedures Act 1986 and with an internal ethics board and
UK government approved project (P63FE629C) and personal license (IC37CBB8F).
Food and water were supplied ad libitum. Female BALB/c
mice (Charles River, UK) 6–8 weeks of age were placed into
groups (n = 5) and housed in a fully acclimatized
room. In vivo imaging was performed as previously
described.43 (link) Mice were injected either
intramuscularly in both hind legs or intradermally with 5 μg
of fLuc saRNA complexed with either pABOL or PEI in a total volume
of 50 μL. After 7 days, the mice were injected intraperitoneally
(IP) with 100 μL of XenoLight RediJect
substrate (PerkinElmer, UK) and allowed to rest for 10 min. Mice were
then anesthetized using isoflurane and imaged on an In Vivo Imaging System (IVIS) FX Pro (Kodak Co., Rochester, NY, USA) equipped
with Molecular Imaging software version 5.0 (Carestream Health, USA)
for 2 min. Signal from each injection site was quantified using Molecular
Imaging software and expressed as relative light units (p/s).
7
Luciferase Reporter Gene Expression
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All animals were handled in accordance with the UK Home Office Animals Scientific Procedures Act 1986 and with an internal ethics board (the Animal Welfare and Ethical Review Body (AWERB)), and UK government approved project license (P63FE629C) and personal license (IC37CBB8F). Food and water were supplied ad libitum. Female BALB/c mice (Charles River, UK) 6–8 weeks of age were placed into groups (n = 5) and housed in a full acclimatized room. In vivo imaging was performed as previously described [5 (link)]. Mice were injected intramuscularly (IM) in both hind leg quadriceps with 5 μg of fLuc saRNA formulations in a total volume of 50 μL. After 7 days, the mice were injected intraperitoneally (IP) with 150 μL of XenoLight RediJect™ D-Luciferin substrate (PerkinElmer, UK) and allowed to rest for 10 min. Mice were then anesthetized using isoflurane and imaged on an In Vivo Imaging System (IVIS) FX Pro™ (Kodak Co., Rochester, NY, USA) equipped with Molecular Imaging software version 5.0 (Carestream Health, USA) for 2 min. The signal from each injection site was quantified using Molecular Imaging software and expressed as total flux (p/s).
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