Total RNA was extracted from HeLa cells using QIAzol Lysis Reagent (Qiagen SpA, Milan, Italy), and reverse transcription was performed according to Laforenza et al. [62 (link)]. cDNA amplification was performed by GoTaq® Flexi DNA Polymerase (Promega, Milano, Italy), as previously described [62 (link)]. The primers used for amplification are listed in the Table 2 . The PCR protocol consisted of an initial denaturation of 3 min at 95 °C followed by 35 cycles of denaturation at 96 °C for 30 s, annealing (see TM in Table 1 ) for 30 s, and extension at 72 °C for 30 s. Reverse transcription was always performed both either in the presence (positive) or in the absence (negative control) of reverse transcriptase enzyme. PCR products were separated on a 3% Nusieve® (2:1) gel agarose, stained with ethidium bromide, and acquired with the Image Master VDS (GE Healthcare, Milano, Italy). The molecular weight of the PCR products was compared with the DNA molecular weight marker VIII (Roche Molecular Biochemicals, Monza, Italy).
Image master vds
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden
The Image Master VDS is a laboratory imaging system designed for visualizing and documenting gel-based samples. It provides high-quality digital images of gels, blots, and other samples. The system utilizes a high-resolution camera and specialized software to capture and process the images.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (7 mentions)
Hrp conjugated immunoglobulin, by Jackson ImmunoResearch (4 mentions)
Extraction kit, by Beyotime (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Hrp conjugate immunoglobulin, by Jackson ImmunoResearch (2 mentions)
One step rt pcr kit, by Takara Bio (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Dna molecular weight marker 8, by Roche (1 mentions)
Gotaq flexi dna polymerase, by Promega (1 mentions)
Wizard genomic purification kit, by Promega (1 mentions)
Suprimescript rt premix, by GeNet Bio (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Prime taq premix, by GeNet Bio (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Nanodrop, by Mecasys (1 mentions)
Ckx41, by Nikon (1 mentions)
Ds u3, by Nikon (1 mentions)
Matrigel, by BD (1 mentions)
Monoclonal anti β actin, by Cell Signaling Technology (1 mentions)
Monoclonal anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
30 protocols using image master vds
1
Reverse Transcription and PCR Analysis of HeLa Cells
2
Quantifying MAPC Cells via PCR
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Detection of MAPC was performed with a PCR for human β-2 microglobulin [2 (link)]. Genomic DNA was isolated from snap frozen brain, spleen, and lung tissues using the Wizard Genomic Purification Kit (Promega, Leiden, The Netherlands) according to the manufacturer’s recommendations. PCR reactions of genomic DNA were performed for β-2 microglobulin followed by a nested PCR for β-2 microglobulin on the amplification product of the first PCR.
The amplified products were separated on ethidium bromide-stained 2 % agarose gels and captured using the Imagemaster® VDS equipped with a CCD camera (GE Healthcare Life Sciences (Pharmacia Biotech), Uppsala, Sweden). Serial dilutions of MAPC cells were used as references.
Primers used for the amplification of β-2 microglobulin were 5′- GTGTCTGGGTTTCATCAATC-3′ (sense), 5′- GGCAGGCATACTCATCTTTT-3′ (antisense), 5′- TGGGTTTCATCAATCCGACAT-3′ (nested sense) and 5′- CTCATCTTTTTCAGTGGGGGT-3′ (nested antisense).
The amplified products were separated on ethidium bromide-stained 2 % agarose gels and captured using the Imagemaster® VDS equipped with a CCD camera (GE Healthcare Life Sciences (Pharmacia Biotech), Uppsala, Sweden). Serial dilutions of MAPC cells were used as references.
Primers used for the amplification of β-2 microglobulin were 5′- GTGTCTGGGTTTCATCAATC-3′ (sense), 5′- GGCAGGCATACTCATCTTTT-3′ (antisense), 5′- TGGGTTTCATCAATCCGACAT-3′ (nested sense) and 5′- CTCATCTTTTTCAGTGGGGGT-3′ (nested antisense).
3
RNA Extraction and qPCR from RAW 264.7 Cells
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For total RNA extraction, RAW 264.7 cells were pre-treated with 6FU (25 and 50 µM) prior to stimulation with LPS (1 µg/ml), subsequently RNA was extracted with 2-mercaptoethanol the and RNeasy plus mini kit, according to the manufacturer's protocol (Qiagen GmbH, Hilden, Germany). The concentration of total RNA was measured using a nanodrop (Mecasys Co., Ltd., Daejeon, Korea) and 2 µg RNA was synthesized to cDNA using a SuPrimeScript RT Premix (GeNet Bio, Inc., Daejeon, Korea) under the following conditions; 50˚C for 60 min and 70˚C for 10 min. The cDNA was amplified using Prime Taq Premix (GeNet Bio, Inc.) with specific primers presented in Table I, according to the manufacturer's protocols (95˚C for 30 sec, 55˚C for 30 sec, and 72˚C for 30 sec, 30 cycles). Amplified PCR products were stained with ethidium bromide (Sigma-Aldrich; Merck KGaA) and visualized in a 2% agarose gel using ImageMaser ® VDS Software version 3.0 in ImageMaster ® VDS GE Healthcare, Chicago, IL, USA).
4
Densitometric Analysis of Proteins
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Image Master® VDS (Pharmacia Biotech Inc., San Francisco, CA) was used for scanning densitometry. All results are representative of three independent experiments performed in triplicate. Significant differences within experiments were evaluated by one-way analysis of variance and the Scheffe post-hoc test. P-values less than 0.05 were treated as statistically significant.
5
Matrigel Invasion Assay for Breast Cancer Cells
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The upper chambers of the inserts were coated with 100 µl of Matrigel (1 mg/ml, BD Bioscience). Control or P2Y 2 R siRNA-transfected breast cancer cells (2x10 5 cells/insert) were added to the upper chambers in serum-free media and 500 µl of RPMI media with or without apyrase was added to the lower chambers. The cells were incubated for 16 h to facilitate invasion, and subsequently the cells on the lower part of the insert membranes were stained with 4' ,6-diamidino-2-phenylindole (DAPI) and counted in a 500x500 µm field under an Olympus microscope (CKX41) equipped with a camera (Nikon, DS-U3). Three independent experiments were performed in triplicate.
Data analysis. Image Master ® VDS (Pharmacia Biotech Inc., San Francisco, CA, USA) was used for scanning densitometry.
All results are representative of three independent experiments performed in triplicate. Significant differences within experiments were evaluated using one-way analysis of variance and the Scheffe post-hoc test. P<0.05 was considered statistically significant.
Data analysis. Image Master ® VDS (Pharmacia Biotech Inc., San Francisco, CA, USA) was used for scanning densitometry.
All results are representative of three independent experiments performed in triplicate. Significant differences within experiments were evaluated using one-way analysis of variance and the Scheffe post-hoc test. P<0.05 was considered statistically significant.
6
Quantifying MMP-2/MMP Gelatinolytic Activity
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MMP-2/MMP zymography from the blood samples was conducted. Gelatinolytic activity appeared as a clear band over a blue background. Using an Image Master VDS (Pharmacia Biotech), images taken with the same magnification were quantified by densitometry based on their contour quantity after background subtraction. Arbitrary densitometry units were correlated with the standard curve prepared by serial dilution of human recombinant gelatinases across a linear range (0.030–1.25 ng/ml). Seventy-six BAL samples in total were selected for examination and analysis [44 (link)–50 (link)].
7
Western Blot Analysis of Cell Signaling Proteins
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Extraction kit (Beyotime, Shanghai, China) was used to isolate total-cell extracts and nuclear extracts. For all samples, 20 μg cell lysate was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the separated proteins were transferred into a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Blots were probed with anti-GLI1, anti-GAL-1, anti-MMP14, anti-MMP2, anti-LAMC2 or anti-GAPDH primary antibody at a dilution of 1:2,000. The secondary HRP antibody was also used at a dilution of 1:2,000. Protein bands were visualized using West Picochemiluminescent Substrate (Pierce, Carlsbad, CA, USA) and quantified using densitometric image analysis software (Image Master VDS; Pharmacia Biotech). GAPDH was used as a loading control. All western blotting analysis assays were performed in triplicate.
8
Western Blot Analysis of Apoptosis and Signaling
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Cell lysates were prepared and 20 µg of these were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Specific monoclonal anti-cleaved caspase-3 [Cell Signal Technology (CST), SN: 4380, dilution: 1:2000], monoclonal anti-Bcl-2 (CST, SN: 11988, dilution: 1:2000), monoclonal anti-p65 (CST, SN: 5741, dilution: 1:2000), monoclonal anti-p-p38 (CST, SN: 3195, dilution: 1:2000), and monoclonal anti-β-actin (CST, SN: 8457, dilution: 1:4000) antibodies were used. HRP-conjugated immunoglobulin was used as the secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). West Pico chemiluminescence was used as the substrate to visualize protein bands, which were quantified using densitometric image analysis software (Image Master VDS; Pharmacia Biotech) and normalized to β-actin expression.
9
Protein Expression Analysis Using Western Blot
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Total cell extracts and nuclear extracts were prepared using an extraction kit (Beyotime, Shanghai, China). 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were used to separate 20 μg of cell lysates and the separated proteins were transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Blots were probed with rabbit monoclonal antibody against Gal-1 (Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal antibody against vimentin (Cell Signaling Technology), rabbit monoclonal antibody against anti-E-cadherin (Cell Signaling Technology), or mouse monoclonal antibody against GAPDH (Kang Cheng, Shanghai, China) antibodies at a dilution of 1:2000. Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin and HRP-conjugated goat anti-rabbit immunoglobulin were used as a secondary antibody at a dilution of 1:2000. The West Pico chemiluminescent Substrate (Pierce, Carlsbad, CA, USA) was used to visualize the immunoreactive protein bands, and densitometric image analysis software (Image Master VDS; Pharmacia Biotech) was used to quantify the visualized protein bands. The level of GAPDH was used as an internal reference. All experiments were performed in triplicate.
10
Quantitative Western Blot Analysis
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The proteins extracted from tissues were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the separated proteins were transferred onto a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Blots were probed with anti-TGF-β1, anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3, or anti-GAPDH primary antibodies at a dilution of 1:2,000. The anti-GAPDH antibody (bs-0755R) was provided by Bioss (Beijing, China). The secondary HRP-conjugated antibody was also used at a dilution of 1:2,000. The West Pico chemiluminescent Substrate (Pierce, Carlsbad, CA, USA) was used to visualize the protein bands and densitometric image analysis software (Image Master VDS; Pharmacia Biotech) was used to quantify the protein bands. All assays were performed in triplicate.
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