Human BMP2 and VEGF gene primers were designed and amplified as described previously (11 (link),16 (link)). The oligonucleotides were combined into the lentiviral vector pCDH-CMV-MCS-EF1-copGFP (pCDH; System Biosciences, Mountain View, CA, USA) to build the recombinant plasmids, pCDH-BMP2 and pCDH-VEGF. The recombinant plasmids and packaging plasmids were then co-transfected into 293FT cells (Cyagen Biosciences, Inc.). The media of the 293FT cells containing lentivirus was collected 48 h after transfection, and then were purified by ultracentrifugation at 1,000 × g, 37°C for 10 min, and the supernatant was subsequently filtered through a 0.2-µm syringe filter (EMD Millipore, Billerica, MA, USA). The 4th passage SCAP were infected with the cell supernatants which contained lentiviral constructs (multiplicity of infection =70) to obtain SCAP-BMP2 and SCAP-VEGF. Furthermore, SCAP-BMP2-VEGF were generated by infecting SCAP with cell supernatants which contained lentiviral constructs expressing BMP2 and VEGF, sequentially. Serving as the control, a blank vector transfected with SCAP [SCAP-green fluorescent protein (GFP)] was constructed. The expression of BMP2 and VEGF in the four groups of cells was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis four days subsequent to transfection.
0.2 m syringe filter
Manufactured by Merck Group
Sourced in United States
The 0.2-μm syringe filter is a laboratory equipment designed to remove particulates and microorganisms from liquids. It features a 0.2-micron pore size membrane that effectively traps contaminants while allowing the desired liquid to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Pcdh cmv mcs ef1 copgfp pcdh, by System Biosciences (1 mentions)
293ft cells, by Cyagen (1 mentions)
Nh4 2so4, by Thermo Fisher Scientific (1 mentions)
Ktaprime plus chromatography system, by GE Healthcare (1 mentions)
Hitrap protein g hp column, by GE Healthcare (1 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
6 protocols using 0.2 m syringe filter
1
Lentiviral Transduction of SCAP with BMP2 and VEGF
2
Herbal Decoction for Therapeutic Applications
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YCHD consists of Artemisiae scopariae herba (27 g), Gardeniae Fructus (6 g), and Radix et Rhizoma Rhei (9 g) (28 (link)). The medicinal herbs were obtained from the Chinese Pharmacy at Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. The process of extraction was performed according to a previous published patent (patent no. ZL201010501560.7) and article (29 (link)). Briefly, herbs were soaked in distilled water (1:10, w/v) for 2 h at 20˚C and then boiled twice for 30 min at 100˚C. The twice-boiled solutions were blended and filtered through a 0.2 µm syringe filter (EMD Millipore). Finally, the filtered liquids were concentrated under pressure at 60˚C and stored at 4˚C. tauroursodeoxycholic acid (TUDCA) is a water-soluble bile acid that is commonly used in clinical settings (30 (link)) and was provided by Bruschettini S.R.L. TUDCA was used as the positive control drug in the present study.
3
Purification of Novel Mouse Monoclonal Antibodies
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Isotyping of novel mAbs was determined using a mouse mAb isotyping kit (AbD Serotec, UK) according to the manufacturer’s protocol and the antibodies were purified by affinity chromatography as described previously9 (link). Briefly, novel mouse mAbs were purified by salt fractionation (solid ammonium sulphate ([NH4]2SO4; 45% of saturation − 270 g/L; Fisher Scientific) followed by affinity chromatography using a 5 ml HiTrap Protein G HP column in an ÄKTAprime plus chromatography system (GE Healthcare, UK), as described previously9 (link). The purified antibodies were filtered through a 0.2 µm syringe filter (Merck Millipore, UK), aliquoted and stored at −20 °C for further studies.
4
Extraction and Concentration of Helminth Excretory-Secretory Products
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4hr and overnight supernatants containing E/S products from passage worm cultures were filter sterilised through a 0.2µm syringe filter (Merck, Germany). E/S was then concentrated using Amicon Ultra-15 centrifugal filter units (Merck) by centrifugation at 3,000g at 4°C. Centrifugation was carried out until approximately 20% of the original volume of supernatant remained. E/S was then dialysed against PBS using Slide-A-Lyzer Dialysis cassettes, 3.500 MWCO (Thermo Scientific, UK) at 4°C. The E/S was then filtered sterilised a final time through a 0.2µm syringe filter, the protein concentration measured and aliquoted prior to storage at -80°C
5
Chlorsulfuron Quantification in Water
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Ethylenediaminetetraacetic acid (EDTA) was added to the tap water and environmental water samples to a final concentration of 1 mM to remove any heavy metal ions. Then, the solution was filtered through a 0.2 µm syringe filter (Millipore). The prepared suspension was spiked with standard solutions at concentrations of 0.5, 1.0, and 5.0 mg/L. Finally, the colorimetric method was used to determine the chlorsulfuron concentration.
6
Differential Scanning Calorimetry of Purified Ligases
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Differential scanning calorimetry (DSC) experiments were carried out using an N-DSC III differential scanning calorimeter (Calorimetry Sciences Corporation). Purified ligases with concentrations of 1-2 mg ml -1 were extensively dialyzed against 50 mM HEPES pH 8.0, 100 mM NaCl to ensure complete equilibration. The enzymes were filtered through a 0.2 µm syringe filter (Millipore, Billerica, USA) and degassed for approximately 15 min before being loaded into the sample cell. The dialysis buffer was used as reference for baseline subtraction. Data analysis was performed using the program NanoAnalyse 2.4 (TA instruments). For each protein sample scanned the corresponding buffer baseline was subtracted, and the data were normalized to the molar protein concentration calculated from the absorbance at 280 nm after dialysis and filtration. The calorimetric enthalpy was determined directly from the experimental data, and a theoretical two-state model was fitted using the routines provided in the program for determination of the van 't Hoff enthalpy.
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