Cells were seeded in 6-well plates (3 × 105 cells/well). The next day, the cells were trypsinized and centrifuged for 5 min at 300 g. The supernatant was discarded, and the pellet was resuspended in 350 μL RLT buffer (Qiagen) with 1% BME (M6250, Sigma-Aldrich). The samples were then homogenized using QIAshredder (Qiagen), and the total RNA was purified with an RNeasy Minikit (Qiagen), following the manufacturer’s instructions. cDNA was then produced using GoScript Reverse Transcriptase (Promega), following the manufacturer’s instructions. Target loci (using the primers listed in Table 1 ) were amplified using CloneAmp HiFi PCR Premix (TaKaRa), following these steps: (i) 98°C for 30 s; (ii) 30 cycles at 95°C for 10 s, 55°C for 5 s, and 72°C for 35 s; and (iii) 72°C for 2 min. The PCR product was then stained with TriTrack DNA Loading Dye 6× (Thermo Fisher Scientific), and electrophoresis was performed using 1% agarose gel. Fluorescent images were taken with a Quantum imaging device, using the VisionCapt software (Vilber Lourmat).
Vision capt software
Manufactured by Vilber
Sourced in France
Vision Capt software is a digital imaging and analysis tool designed for laboratory applications. It captures, processes, and analyzes images obtained from various imaging devices. The software provides core image processing and measurement functionalities.
Automatically generated - may contain errors
Lab products found in correlation
Ebox vx5, by Vilber (2 mentions)
Qiashredder, by Qiagen (1 mentions)
Goscript reverse transcriptase, by Promega (1 mentions)
Cloneamp hifi pcr premix, by Takara Bio (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Mini protean 2 unit, by Bio-Rad (1 mentions)
Molecular protein mass marker, by Bio-Rad (1 mentions)
Mini protean 2, by Bio-Rad (1 mentions)
Protein molecular weight marker, by Bio-Rad (1 mentions)
Mtt assay, by Roche (1 mentions)
Proteinase and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Affinipure goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
6 protocols using vision capt software
1
RNA Extraction and cDNA Synthesis Protocol
2
SDS-PAGE Protein Separation Protocol
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The SDS-PAGE was performed according to a method described by Schägger and Von Jagow [55 (link)] 4% stacking gel (w/v) and 12% polyacrylamide gel (w/v). 10 milligrams of protein isolate was dissolved in 1 mL of denaturant sample buffer (0.5 M Tris-HCl pH 6.8, glycerol, 10% SDS, w/v, 0.5% bromophenol blue, w/v, β-mercaptoethanol), and heated at 95 °C. Then, 10 µL of the sample was loaded to the sample wells. Protein separation was carried at 80 V for 30 min followed by 110 V for 90 min for the resolving gel using a Mini Protean II unit (Bio-Rad Laboratories, Hercules, CA, USA). The gel was stained for 40 min with Brilliant Blue (Bio-Rad, Coomassie R250). Destaining of the gel was done three times using water/methanol/acetic acid (7/2/1, v/v/v) for 15 min each cycle with shaking using an orbital shaker (Fristek S10, Taichung city, Taiwan). Estimation of the molecular mass of proteins was done using molecular protein mass marker (250 to 10 kDa, Bio-Rad) loaded at 5 uL in the sample well. The gels was scanned with E-Box VX5 (Vilber Lourmat, Paris, France) and the analysis of the captured image was done using Vision Capt software (V16.08a, Vilber Lourmat, Paris, France).
3
SDS-PAGE Protein Separation and Analysis
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SDS-PAGE was performed according to the method described by Schägger and Von Jagow [28 (link)] using 4% stacking gel (w/v) and 12% polyacrylamide gel (w/v). First, 10 milligrams of protein isolate was dissolved in 1 mL of denaturing buffer for samples, (0.5 M Tris-HCl pH 6.8, glycerol, 10% SDS, 0.5% bromophenol blue, β-mercaptoethanol) and heated at 95 °C. Then, 10 μL of the sample was loaded into the sample wells. Protein separation was performed at 80 V for 30 min, then at 110 V for 90 min for a separation gel using a Mini Protean II device (Bio-Rad Laboratories, Hercules, CA, USA). The gel was stained with brilliant blue for 40 min (Bio-Rad Coomassie R250, Bio-Rad Laboratories, Hercules, CA, USA). Gel bleaching was performed three times using water/methanol/acetic acid (7/2/1 v/v/v) for 15 min each shaking cycle using an orbital shaker (Fristek S10, Taichung, Taiwan). The molecular weight of proteins was evaluated using a protein molecular weight marker (250 to 10 kDa, Bio-Rad Laboratories, Hercules, CA, USA) loaded at a dose of 5 μL into the sample well. Gels were scanned with an E-Box VX5 (Vilber Lourmat, Paris, France), and captured image analysis was performed using Vision Capt software (V16.08a, Vilber Lourmat, Paris, France).
4
Clonogenic Survival and Apoptosis Assays
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For clonogenic survival analysis, 5×103 A431 or Difi cells were seeded in 6-well plates in the presence of the indicated antibodies or pharmacological inhibitors. Following incubation for 7 to 14 days colonies were fixed with ethanol (70% v/v) for 30 minutes, stained with Coomassie brilliant blue and automatically counted using an Infinity-100 System and the Vision Capt software (Vilber Lourmat, Eberhardzell, Germany). Proliferation was quantified by means of the MTT assay according to the manufacturer's instruction (Roche, Mannheim, Germany). Apoptosis was quantified by flow cytometric determination of cells with subgenomic DNA content following hypotonic lysis and staining with propidium iodide as previously described [44 (link)]. All results were obtained from at least three independent experiments.
5
Protein Separation by SDS-PAGE
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C. angulata proteins were separated through SDS-PAGE as described by Laemmli [48 (link)]. Firstly, 1 mg of sample (dry weight, protein basis) was diluted in 1 mL sample buffer [0.5 M Tris–HCl (pH 6.8), glycerol, 10% (w/v) SDS, 0.5% (w/v) bromophenol blue, and β-mercaptoethanol]. The solution was then heated at 95 °C for 4 min and centrifuged at 4000× g for 15 min prior to loading. Electrophoresis was performed in a 12% running gel (ddH2O, 30% Acrylamide/Bis (37.5:1), 1.5 M Tris-HCl (pH 8.8), 10% (w/v) SDS, 10% (w/v) ammonium persulfate and TEMED) and 4% stacking gel (ddH2O, 30% Acrylamide/Bis (37.5:1), 0.5 M Tris-HCl (pH 6.8), 10% (w/v) SDS, 10% (w/v) ammonium persulfate and TEMED). Ten (10) µL of sample and 5 µL of standard (AccuRuler RGB prestained protein ladder, MaestronGen Inc., Taiwan) were loaded into each well of the gel. The voltage of power supply was set at 70 V for stacking gel and 110 V for running gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue for 30 min and subsequently destained with water/methanol/acetic acid (7/2/1, v/v/v) solution for 15 min with continuous shaking. The gel was then scanned using a gel image scanner and the MW of the visible bands was determined using the VisionCapt software (V16.08a, Vilber Lourmat, Paris, France).
6
Muscle Protein Extraction and Western Blot
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Muscle tissues were homogenized in lysis buffer containing 1 mM dithiothreitol, 5 mM EDTA, 10 mM 4-2-hydroxyethyl-1-piperazineethanesulfonic acid, 90 mM KCl, 3 mM MgCl 2 , 0.04% sodium dodecyl sulfate, 1% glycerol, and a proteinase and phosphatase inhibitor cocktail (1:100, Sigma-Aldrich). Whole muscle lysates were centrifuged, the supernatant was collected, and protein was quantified using the Bradford assay (Bio-Rad). 16 Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Bio-Rad), and the membranes were incubated with primary antibodies. After that, the membranes were washed in Tween tris-buffered saline solution (50 mM tris-HCl, pH 7.4, 0.1% Tween 20, and 0.5 M NaCl), incubated with secondary antibodies, and washed in Tween tris-buffered saline solution. Signals were detected using a Fusion-FX image acquisition system and an enhanced chemiluminescence system (Westar ECL-sun, Cyanagen, Italy), and the acquired images were analysed using Vision-Capt software (Vilber Lourmat). 16 The rabbit primary antibodies used were anti-atrogin-1 (1:1,000; #AP2041, ECM Bioscience), anti-Foxo3a (1:1000; #Ab47409, Abcam), and anti-GAPDH (1:2000; #ABS16; Millipore). The secondary antibody used was peroxidase conjugated AffiniPure goat anti-rabbit IgG (1:10,000; #111-035-003, Jackson ImmunoResearch Laboratories).
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