ECM production assays were performed as previously described [24 (link)]. Briefly, fibroblasts were seeded onto 96-well, optical, flat-bottom, black microplates (Nunc, Fisher Scientific) at 5x 103 cells/well. After overnight attachment, they were incubated with or without 10 ng/ml TGF-ß1 in presence or absence of additional compounds for 7 days. Cells were incubated with DRAQ5 for 5 minutes and cell number was determined using an infrared imaging system (Odyssey CLx imager; LI-COR) at 700 nm. Cells were lysed using 0.25 M ammonium hydroxide and ECM was fixed with a solution containing 50% methanol and 7.5% acetic acid for 1 h at–20°C. Afterwards, ECM was stained with Coomassie Blue overnight at 4°C. Plates were scanned using an infrared imaging system (Odyssey CLx imager; LI-COR) at 700 nm.
Odyssey clx imager
Manufactured by LI COR
Sourced in United States, Germany, United Kingdom
The Odyssey CLx Imager is a high-performance infrared imaging system designed for Western blot and multiplex fluorescent assay detection. It provides sensitive and quantitative detection of near-infrared fluorescent proteins and dyes.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (31 mentions)
Image studio software, by LI COR (18 mentions)
Image studio lite software, by LI COR (18 mentions)
Protease inhibitor cocktail, by Roche (18 mentions)
Ripa buffer, by Thermo Fisher Scientific (18 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Image studio, by LI COR (14 mentions)
Irdye 680rd, by LI COR (12 mentions)
Irdye 800cw, by LI COR (12 mentions)
Irdye secondary antibody, by LI COR (12 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Dc protein assay kit, by Bio-Rad (9 mentions)
β actin, by Merck Group (9 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (9 mentions)
Immobilon fl pvdf membrane, by Merck Group (8 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (8 mentions)
Nitrocellulose membrane, by Bio-Rad (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (7 mentions)
Pvdf membrane, by Bio-Rad (7 mentions)
378 protocols using odyssey clx imager
1
Quantitative Extracellular Matrix Production Assay
2
Quantitative Western Blot and Dot Blot
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Transfected HEK293Ts were homogenised in lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS, and 0.5% sodium deoxycholate supplemented with cOmplete protease inhibitor cocktail [Roche]) and sonicated in a Bioruptor. Samples were centrifuged at 16,000g for 10 min. Supernatants were loaded with NuPAGE LDS sample buffer and DTT, then heated to 70°C for 5 min. Samples were separated on NuPAGE 4–12% Bis-Tris gels in MES running buffer, then transferred onto PVDF membranes. After blocking, membranes were incubated with anti-FLAG (F3165, 1:4,000; Sigma-Aldrich) or anti-GAPDH (2118S, 1:1,000; Cell Signaling Technologies) followed by complementary secondary antibodies (LI-COR IRDye, 1:10,000). Specific binding was detected with a LI-COR Odyssey CLx imager. The intensity of bands was quantified using Fiji–ImageJ software. Statistical analyses were performed using GraphPad Prism 9. Details are given in the figure legend. For dot blotting, supernatants of centrifuged samples prepared as above were dotted onto a nitrocellulose membrane (18 or 9 μg of total protein per dot). After blocking, membranes were incubated with anti-FLAG (F3165, 1:400; Sigma-Aldrich) or anti-GAPDH (2118S, 1:400; Cell Signaling Technologies) followed by complementary secondary antibodies (LI-COR IRDye, 1:10,000). Specific binding was detected with a LI-COR Odyssey CLx imager.
3
Protein Expression Analysis in Liver Cancer Cells
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Liver cancer cells were collected, then incubated with lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology) on ice for 30 min. The protein concentrations were determined by the BCA method using Enhanced BCA Protein Assay kit (cat. no. P0009; Beyotime Institute of Biotechnology). Proteins (40 µg) were separated by SDS-PAGE on 8-12% Tris-glycine gels. Following electrophoresis, the proteins were transferred to PVDF membranes, which were then blocked with 5% skimmed milk for 2 h at room temperature followed by washing with TBS + 0.1% Tween-20 (TBST) three times. Following incubation with primary antibodies overnight at 4°C, the membranes were washed thrice with TBST and then incubated with the corresponding secondary antibodies (DyLight™ 800 4X PEG-conjugated anti-rabbit-IgG; DyLight™ 800 4X PEG-conjugated anti-mouse-IgG; 1:50,000; Cell Signaling Technology) for 2 h at room temperature. Finally, after washing three times with TBST, 5 min each, the bands were visualized using an LiCor Odyssey CLx imager and Odyssey CLX Image Studio software version 5.0.21 (LI-COR Biosciences) (17 (link)). ImageJ (version: 1.52a; National Institutes of Health) was used to semi-quantitatively analyze protein expression levels.
4
Quantifying Active β-Catenin Signaling
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Cells were lysed in ice-cold RIPA buffer and protein concentrations were determined using the DC Protein Assay Kit (BioRad) following manufacturer’s instructions. Incubation with primary antibodies against active β-catenin (1:1000, BD Sciences, Franklin Lakes, NJ, USA) and β-actin or GAPDH (1:5000, Thermo Fisher) was performed overnight at 4 °C on a 3D-shaker in 5% BSA (VWR Life Science, Radnor, PA, USA) in TBST. As secondary antibodies, we used goat-anti-rabbit antibody IRDye800CW (1:10,000, LI-COR #926-32211, Lincoln, NE, USA) and goat-anti-mouse antibody IRDye680RD (1:10,000, LI-COR #926-68070) diluted in blocking solution and incubated for 1 h at room temperature. Signal detection was performed on a luminescence-based system in a LI-COR Odyssey CLx Imager (LI-COR). Luminescence values for active β-catenin were normalized to the corresponding GAPDH or β-actin values.
5
Western Blot Analysis of EMT Markers
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Cells were lysed in ice-cold RIPA buffer and protein concentrations were determined using the DC Protein Assay Kit (BioRad). Incubation with primary antibodies against ZEB1 (1:2000, Sigma #HPA027524), CHKα (1:500, Abcam #ab88053), TWIST1 (1:100, Santa Cruz #sc-81417), β-actin (1:1000, Santa Cruz #sc-130657) and α-tubulin (1:10000, Sigma #T9026) was performed overnight at 4°C on a 3D-shaker in 5% milk powder (Carl Roth) in TBST. As secondary antibodies we used goat-anti-rabbit IRDye800CW (1:10000, LI-COR #926-32211), goat-anti-mouse IRDye680RD (1:10000, LI-COR #926-68070) and goat anti-rabbit-HRP (1:10000, Jackson Immuno Research #111-035-144) diluted in blocking solution and incubated for 1 h at room temperature. Signal detection was performed either on a film based system by applying Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific) or on a luminescence based system in a LI-COR Odyssey CLx Imager (LI-COR). Densitometry was done using supplied software from LI-COR or ImageJ software [75 (link)]. Densitometry values for ZEB1 and CHKα were normalized to the corresponding alpha tubulin values, TWIST1 was normalized to beta actin.
6
Quantifying Protein Expression in KO Cells
7
Glioma Cell Protein Extraction and Analysis
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Total proteins were extracted from glioma cells using RIPA Buffer as reported. Protein concentrations were determined in a Tecan Safire 2 Multiplate reader (Tecan) using the DC Protein Assay Kit (Biorad) due to the manufacturer´s instructions. Primary antibodies (ALDH3A1, 1/1000, abcam # ab76976; β-catenin, 1/1000, BD # 610153; α-Tubulin, 1/10000, Sigma # T9026; CD133, 1/100, Miltenyi Biotec # W6B3C1) were incubated overnight at 4°. Secondary antibodies (goat-anti-rabbit, IRDye800CW LI-COR # 926-32211; goat-anti-mouse, IRDye680RD LI-COR # 926-68070; goat anti-rabbit-HRP, Jackson Immuno Research # 111-035-144; all 1/10000) were incubated 1h at room temperature. All antibodies were diluted in blocking solution containing 5% milk powder in Tris-buffered saline with Tween20 (TBST). Signals were detected using a film based system by applying Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific) or a luminescence based system in a LI-COR Odyssey CLx Imager (LI-COR). Densitometry was done using supplied software from LI-COR or ImageJ software.
8
Protein Extraction and Analysis from Esophageal Biopsies
9
Western Blot Analysis of SARS-CoV-2 Proteins
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HEK293T or VeroE6-APN cells were grown in 6-well plates and transfected with the pCAGGS_ORF3-FL, pCAGGS_ORF3-Trnc or pCAGGS_SAV12 using Fugene HD transfection reagent (Promega, Madison, WI, USA) following the manufacturer’s instructions. At 24 h post transfection (hpt), transfected cells were collected and re-suspended in mammalian cell lysis buffer. Fifty micrograms of protein samples were incubated with protein loading buffer at 37 °C for 1 h, loaded on to 10% polyacrylamide gel, and transferred to nitrocellulose membranes. Membranes were then probed with rabbit anti-myc antibodies (Abcam, Cambridge, MA, USA) and followed by IRDye® 800CW goat anti-rabbit IgG (Li-COR bioscience, Lincoln, NE, USA) antibodies. Protein bands were visualized using a Li-COR Odyssey CLx imager (Li-COR bioscience).
10
Western Blot Protein Quantification Protocol
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Cell pellets were collected and lysed (mammalian PER, Thermo scientific + 1x protease inhibitor cocktail, Thermo Scientific), and samples were prepared after protein quantification. We loaded about 15 µg protein per lane of a polyacrylamide gel (NuPAGE™ Novex™ 4–12% Bis-Tris Protein Gels). Once the gels were resolved, they were transferred onto nitrocellulose membrane and subsequently blocked in 5% milk solution for a minimum of 2 h. This was followed by a one-step i-Bind process which treated the membrane with primary antibody, washing and secondary antibody steps (Life technologies). We employed LiCor® IRDye secondary antibodies (680 and 800 wavelength infrared dyes) and detection of bands was carried out in a LiCor ODyssey CLx imager (LiCor). Antibodies and dilutions employed can be found in Supplementary Table 3 . Full blots are provided in Supplementary Figs. 4 and 5 .
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