The cells with different treatments were lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail (Roche Applied Science). Twenty micrograms of total protein from sEVs lysate were loaded and separated on a standard sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies to the NETs signaling molecules, followed by peroxidase-conjugated secondary antibodies. The results were detected using a charge-coupled device (CCD) camera-based imager (GE Healthcare Life Sciences, Chicago, IL, USA) after membrane incubation with enhanced chemiluminescence (ECL) substrates (Millipore). The protein levels of NETs signaling molecules were normalized to β-actin.
Ccd camera based imager
Manufactured by GE Healthcare
Sourced in United States
The CCD (Charge-Coupled Device) camera-based imager is a lab equipment product designed for image capture and analysis. It utilizes a CCD sensor to convert light energy into electrical signals, enabling high-quality digital image acquisition. The core function of this imager is to provide reliable and precise image data for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride pvdf membrane, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Enhanced chemiluminescence ecl substrate, by Merck Group (2 mentions)
Ecl substrate, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Nuclear extraction kit, by Abcam (1 mentions)
Bca assay, by Abcam (1 mentions)
Gm130, by Cell Signaling Technology (1 mentions)
Flotillin 1, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
9 protocols using ccd camera based imager
1
Quantification of NET Signaling Molecules
2
Protein Expression Analysis of Engineered Breast Cancer Cells
3
Protein Expression Analysis of Exosomes
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The cells with different treatments were lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) containing a protease inhibitor cocktail (Complete, Roche, Germany). Twenty micrograms of total protein from exosome lysate were loaded and separated on a standard sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were incubated with primary antibodies, followed by peroxidase-conjugated secondary antibodies. The results were detected using a charge-coupled device (CCD) camera-based imager (GE Healthcare Life Sciences, USA) after membrane incubation with enhanced chemiluminescence (ECL) substrates (Millipore, USA). The levels of specific protein were normalized to β-actin. The ImageJ software was used for image acquisition and densitometric analysis of the immunoblots. All results were obtained in three independent experiments and the data are presented as the mean ± SD. An unpaired, two-tailed Student’s t-test was performed for between-group comparisons using GraphPad Prism software version 8. All results of densitometric analysis are presented in Additional file 2 .
4
Western Blot Protein Analysis Protocol
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The cells with different treatments were lysed in a radio-immunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) that contained a protease inhibitor cocktail (Complete, Roche, Germany). 20 μg of total protein from an exosome lysate were loaded and separated on a standard sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were incubated with primary antibodies, and this was followed by peroxidase-conjugated secondary antibodies. The results were detected using a charge-coupled device (CCD) camera-based imager (GE Healthcare Life Sciences) after membrane incubation with enhanced chemiluminescence (ECL) substrates (Millipore).
5
Western Blot Protein Analysis
6
Protein Expression Analysis of Engineered Breast Cancer Cells
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E0771, E0771-dCas9-VP64, and E0771-dCas9-VP64-MPH cells in 6-well plates were washed twice with ice cold PBS before lysis with 1× RIPA buffer which was kept on ice for 15 min. Cell lysates were centrifuged at 12,000 g for 15 min at 4°C and protein-containing supernatant was collected. Protein concentration was measured using a standard Bradford assay (BioRad) and 20 μg of protein in each sample were loaded into SDS-PAGE gel. After electrophoresis, proteins separated in gel were transferred into nitrocellulose membranes. Membranes were blocked at room temperature for 1 h using 5% skim-milk in TBST, followed by the incubation with primary antibody in 4°C overnight. After washing three times with TBST, horseradish peroxidase (HRP)-conjugated secondary antibody was added and incubated at room temperature for 30 – 60 min. The chemiluminescent substrate (Clarity Western ECL Substrate, Bio-Rad) was added on top of blot membrane according to the manufacturer’s instructions. The signals were captured using a CCD camera-based imager (GE Healthcare).
7
Western Blot Analysis of Exosome Lysates
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The cells with different treatments were lysed in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) containing a protease inhibitor cocktail (Complete, Roche, Germany). Twenty micrograms of total protein from exosome lysate was loaded and separated on a standard SDS-polyacrylamide gel electrophoresis (PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were incubated with primary antibodies, followed by peroxidase-conjugated secondary antibodies. The results were detected using a charge-coupled device (CCD) camera-based imager (GE Healthcare Life Sciences, USA) after membrane incubation with enhanced-chemiluminescence (ECL) substrates (Millipore, USA).
8
Immunoblotting Analysis of Exosomal Proteins
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The cells with different treatments were lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail (Complete, Roche). Twenty micrograms of total protein from exosome lysate were loaded and separated on a standard sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membranes were incubated with primary antibodies, followed by peroxidase-conjugated secondary antibodies. The results were detected using a charge-coupled device (CCD) camera-based imager (GE Healthcare Life Sciences) after membrane incubation with enhanced chemiluminescence (ECL) substrates (Millipore). The levels of specific protein were normalized to β-actin. ImageJ software was used for image acquisition and densitometric analysis of the immunoblots. All results were obtained in 3 independent experiments, and the data is presented as the mean ± SD. An unpaired, two-tailed Student's t test was performed for between-group comparisons using GraphPad Prism software version 8. All results of densitometric analysis were presented in in Supplementary File 1.
9
Extracellular Vesicle Protein Analysis
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