A hematoxylin‐eosin (HE) staining kit, a tartrate‐resistant acid phosphatase (TRAP) staining kit, phosphate‐buffered saline (PBS), and EDTA antigen retrieval buffer (pH 9.0) were obtained from Servicebio. SYBR Green Master Mix was purchased from MedChemExpress. A diaminobenzidine (DAB) color reagent kit was obtained from DAKO. DMEM, antibiotic‐antimycotic solution, fetal bovine serum (FBS), and bovine serum albumin (BSA) were obtained from Gibco. PCR primers and RNase inhibitors were purchased from Sangon Biotech. Matrigel matrix, 96‐well plates and 24‐well Transwell Millipore chambers were purchased from Corning. Antibody information is provided in Table S1 , S2 . Horseradish peroxidase (HRP)‐conjugated secondary antibodies were purchased from Abcam or Servicebio. A highly sensitive ECL detection kit (ready‐to‐use) was purchased from Vazyme. All other chemicals were obtained from Sinopharm unless otherwise indicated.
Sybr green master mix
Manufactured by MedChemExpress
Sourced in United States
SYBR Green Master Mix is a ready-to-use solution containing SYBR Green I dye, DNA polymerase, dNTPs, and necessary reaction buffer components for real-time PCR amplification. The SYBR Green dye binds to double-stranded DNA, allowing for the detection and quantification of DNA targets during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel matrix, by Corning (1 mentions)
96 well plate, by Corning (1 mentions)
Rnase inhibitor, by Sangon (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Wuhan Servicebio Technology (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Edta antigen retrieval buffer, by Wuhan Servicebio Technology (1 mentions)
Tartrate resistant acid phosphatase staining kit, by Wuhan Servicebio Technology (1 mentions)
Pcr primers, by Sangon (1 mentions)
Cfx96 cycler, by Bio-Rad (1 mentions)
Sv total rna isolation system, by Promega (1 mentions)
Bcl 2, by Integrated DNA Technologies (1 mentions)
Bcl2 associated x (bax), by Integrated DNA Technologies (1 mentions)
β actin, by Integrated DNA Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
Takara kit, by Takara Bio (1 mentions)
5 protocols using sybr green master mix
1
Comprehensive Cell Culture and Staining Protocol
2
Heart RNA Extraction and Gene Expression
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Firstly, total RNA was extracted from heart samples using the SV total RNA isolation system (Promega, Madison, USA). Then, the extracted RNA was reversely transcribed into cDNA using a cDNA synthesis kit (MedChemExpress, USA). The amplification was performed in a CFX96 cycler (Bio-Rad, USA) using an RT-PCR kit (Stratagene, USA). Gene expression of Bcl2, BAX, MEF2, and β-actin (Integrated DNA Technologies, Belgium) was detected using SYBR green master mix (MedChemExpress, USA).
3
Quantitative RT-PCR Gene Expression Analysis
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After the isolation of total RNA from indicated cells with TRIzol reagent (Invitrogen) as directed by the manufacturer’s instructions, reverse transcription was performed. Using SYBR GREEN MASTER MIX (Med Chem Express), gene transcripts were quantified by real-time PCR. The ß-actin was used as an internal control. A list of the primers that were used to amplify the target genes was presented in Supplementary Table S1 .
4
Quantifying Liver mRNA Expression Changes
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The total RNA from liver tissues was isolated using the TRIzol method [15 (link)]. The RNA quantity and quality were determined by NanoDrop 8000 (Thermo Fisher Scientific, Waltham, MA, USA), with an OD 260/280 range of ~2. Changes in the mRNA expression of IL-6 and TNF-α in response to DASA with or without NGN treatment were quantified by QuantStudio 6K Flex Real-Time PCR System, using SYBR Green Master Mix (MedChemExpress, Monmouth Junction, NJ, USA). The real-time PCR data were analyzed using the relative gene expression (i.e., 2−ΔΔ CT) method. Briefly, the data were presented as a fold change in gene expression normalized to the endogenous reference gene (GAPDH) using the 2-delta–delta threshold cycle method (2−ΔΔ CT method) [16 (link)].
5
Isolation and Characterization of MDSCs
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In-vivo and in-vitro generated CD11b + Gr1 + MDSCs were sorted on flow cytometry machine and cells pelleted for RNA isolation using RNAiso plus (Takara bio. Inc., Japan). The extracted RNA was converted to cDNA with Takara kit (Takara bio. Inc., Japan). The transcript level of different genes of interest was evaluated via qPCR machine using SYBR green master mix (MedChem Express, USA) and detected on S1.
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