dECM extracts were prepared from CMSC29 and DMSC23. Protein concentrations of each sample were measured using the Pierce protein assay (Thermo Scientific) according to the manufacturer’s protocol with bovine serum albumin as the standard. The protein extracts were resuspended in 7.5 μl XT Sample Buffer supplemented with 1.5 μl XT reducing agent (Bio-Rad), heated to 95°C for 10 mins, and centrifuged at 14,000 g for 2 mins. After centrifugation, each sample was loaded into a 1.0 mm 4–12% gradient Bis-Tris Criterion XT Precast Gel (Bio-Rad) along with Precision Plus protein standard (Bio-Rad). Fibronectin (Life Technologies) and Collagen I (Trevigen, MD, USA) were loaded as high molecular weight protein controls. SDS-PAGE was performed at 150V for 100 mins using XT MOPS buffer (Bio-Rad). The gel was stained with Coomassie Brilliant Blue G-250 overnight and destained in deionized H2O for 1 hour. Visualization of the bands was carried out on a densitometer (GE ImageScanner III) and the image was taken using ImageQuant TL software (GE Healthcare).
Ge image scanner 3
Manufactured by GE Healthcare
Sourced in United States, Germany
The GE Image Scanner III is a diagnostic imaging equipment designed for medical applications. It utilizes advanced imaging technology to capture high-quality images for medical professionals to analyze and diagnose patient conditions. The core function of the GE Image Scanner III is to provide clear and detailed images to support healthcare professionals in their clinical decision-making processes.
Automatically generated - may contain errors
Lab products found in correlation
Criterion xt bis tris precast gel, by Bio-Rad (2 mentions)
Xt sample buffer, by Bio-Rad (2 mentions)
Imagequant tl software, by GE Healthcare (2 mentions)
Xt reducing agent, by Bio-Rad (2 mentions)
Xt mops buffer, by Bio-Rad (2 mentions)
Precision plus protein standard, by Bio-Rad (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Collagen 1, by Bio-Techne (1 mentions)
Mini protean tgx polyacrylamide gel, by Bio-Rad (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Collagen 1, by Merck Group (1 mentions)
Bio safe coomassie, by Bio-Rad (1 mentions)
Precision plus protein ladder, by Bio-Rad (1 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (1 mentions)
Alkaline phosphatase assay kit, by Nanjing Jiancheng (1 mentions)
Image studio lite software, by LI COR (1 mentions)
5 protocols using ge image scanner 3
1
Protein Extraction and SDS-PAGE Analysis
2
Protein Separation and Visualization
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A total of 40 µg of the pre-treated and post-treated CSMPI was added to 2× Laemmli buffer (Bio-Rad, Hercules, CA, USA) with 50 mM dithiothreitol (DTT) and heated at 95 °C for 5 min; then, they were loaded into a 12% pre-cast Mini-PROTEAN® TGX™ polyacrylamide gel (Bio-Rad, CA, USA) for protein separation. The electrophoresis was set at 200 V, and the gel was run for 35 min. Protein bands were visualised using Coomassie Blue stain, and the gel image was captured using the GE Image Scanner III (Ge Healthcare, Chicago, IL, USA).
3
Comparative Analysis of dECM Composition
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SDS-PAGE was used to compare the compositions of the dECMs. Protein extracts were prepared using the Pierce Protein Assay Kit according to manufacturer instructions and resuspended in 7.5 μL XT sample buffer/1.5 μL XT reducing agent (Bio-Rad), placed in a 95°C heating block for 10 min, and centrifuged for 2 min at 14,000 g. Samples were loaded into 1.0 mm 4–12% gradient Bis-Tris Criterion XT Precast Gel (Bio-Rad), with collagen I (Sigma Aldrich) and Precision Plus protein ladder (Bio-Rad) used as controls. Loaded gels were placed into XT MOPS buffer (Bio-Rad) and electrophoresed at 150 V for ~100 min. Gels were then stained with BioSafe Coomassie (Bio-Rad) for at least 2 h and washed with deionized water three times for 5 min each. Bands were visualized using a GE Image Scanner III, with images taken using ImageQuant TL software (GE Healthcare).
4
Quantifying Cellular Alkaline Phosphatase Activity
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CIH group and normoxia group cells were washed by PBS, and then we fixed the cells with paraformaldehyde for 30 minutes at room temperature. Then, we stained the samples with BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) according to manufacturer’s protocol. The images were captured with a scanner (GE Image Scanner III). At the same time, the staining was visualized by a microscope. Ten random fields were chosen to quantify the alp enzyme using Image-J software. The cellular ALP activity was detected using Alkaline Phosphatase Assay Kit (Jiancheng, China). MC3T3-E1 cells were collected with 0.25% trypsin and then lysed in 1% triton on ice for 30 minutes. Then, we mixed 50 µL matrix liquid and 50 µL buffer solution to incubate at 37°C for 15 minutes in 96-well-plates out of light. After that, 150 µL spectrophotometric chromogenic reagent was added into the 96-well plates. Alp activity was measured using a microtiter plate spectrophotometer (Spectrama) at 520 nm.
5
One-dimensional SDS-PAGE Protein Analysis
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A first screening and comparison of protein patterns of all different samples was achieved by one-dimensional SDS-PAGE under reducing conditions on 140 x 140 x 1.5 mm gradient gels (10-15% T, 2.7% C) in a Hoefer SE 600 vertical electrophoresis chamber (Hoefer scientific instruments, San Francisco, CA, USA). For serum samples, 0.5 µl serum were applied per lane and protein bands stained with colloidal Coomassie Blue G-250 (Novex, Invitrogen, Carlsbad, CA, USA) [19] . For the less concentrated fractions from depletion/enrichment experiments, 2.5 μg protein per lane were separated in SDS-PAGE and subjected to silver staining [20] . Gels were scanned in a GE Imagescanner III (GE Healthcare, Munich, Germany). All the images were digitalized and analyzed by Image Studio Lite Software (Li-Cor Biosciences, Lincoln, NE, USA).
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