Chemically inactivated and purified monovalent influenza virus A/Brisbane/59/2007 (H1N1) solution at GMP grade was obtained from Seqirus (Melbourne, Australia) hemagglutinin (HA) (1.6 mg mL−1) determined by Single Radial Immunodiffusion Assay, SRID. Rhodamine B octadecyl ester perchlorate (R18) was purchased from Merck KGaA (The Netherlands) and dissolved in HLPC grade anhydrous ethanol at a final concentration of 10 mM. R18 solution (40 µL) was added to an influenza solution (1 mL) dropwise at room temperature (RT) under continuous stirring at 200 rpm for 15 min. Non-incorporated R18 was removed by separation on a Sephadex G50 column (Merck KGaA). The void volume fraction was collected and further characterized.
Sephadex g 50 column
Manufactured by Merck Group
Sourced in Germany, United States
Sephadex G-50 is a size exclusion chromatography column used for the separation and purification of molecules based on their size. It is a gel filtration medium composed of cross-linked dextran beads. The column is designed to effectively separate molecules with molecular weights ranging from 1,000 to 30,000 daltons.
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Lab products found in correlation
N octyl β d glucopyranoside, by Anatrace (3 mentions)
Chloroform, by Merck Group (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Sonic dismembrator, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Pe 18 0 18 0 peg2000, by Lipoid (1 mentions)
Alw 2 41 27, by Cayman Chemical (1 mentions)
Sk mel 28, by American Type Culture Collection (1 mentions)
Bca protein estimation kit, by Thermo Fisher Scientific (1 mentions)
Trametinib, by LC Laboratories (1 mentions)
Effectene, by Qiagen (1 mentions)
Nanobrook omni, by Brookhaven Instruments (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Millipore membrane, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Toluene, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
23 protocols using sephadex g 50 column
1
Fluorescent Labeling of Influenza Virus
2
PhaC Ap Purification and Characterization
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The reaction mixture consisting of purified rPhaCAp and 100 mM sodium phosphate (pH 7.4) was incubated for 10 min. The reaction mixtures were then loaded onto a Sephadex-G50 column (Merck, Darmstadt, Germany) equilibrated with 100 mM sodium phosphate (pH 7.4). The reaction mixture containing rPhaCAp was eluted with the same buffer at a flow rate of 1 mL/min. The eluted samples were measured for absorbance at 280 nm, and their retention times were compared to those of molecular standards. The standards used for comparison were alcohol dehydrogenase (150 kDa, 18.5 min), BSA (66 kDa, 24.5 min), and ovalbumin (44 kDa, 27.5 min).
3
Transcription Elongation Dynamics Monitoring
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All transcription reactions were performed in 20 mM Tris-HCl pH 8.0, 100 mM KCl, and 20 mM MgCl2. To obtain the transcription complex, the DNA template (450 nM) was incubated for 5 min at 37°C with the E. coli RNAP (900 nM) and σ70 factor (1.4 µM). To initiate transcription elongation, a GUU trinucleotide (10 µM), ATP (2.5 µM), GTP (2.5 µM), and [α-32P] UTP (11 kBq/µL) were added and incubated 10 min at 37°C. Samples were then diluted twice in buffer and were filtered through Sephadex G-50 columns (Sigma-Aldrich). Complete elongation of transcripts was started by the addition of all NTP (25 µM) and heparin (0.9 µg/µL) to allow single round conditions. NusA transcription factor (75 nM) was added to the reaction when required. For each timepoint, a aliquot was taken and the reaction was stopped in chilled formamide (60%). Transcription products were resolved on 8% polyacrylamide gels and quantified using QuantityOne software (Bio-Rad).
4
Preparing Nanobubble Resolvin Nanoparticles
5
Neutrophil-Derived Microparticle Characterization
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Example 2
In some embodiments, the particles described herein can be constructed as follows: Microparticles are generated from isolated human neutrophils following stimulation of the neutrophils. Purity of the microparticles can be assessed by Annexin V and CD66 staining. Organic solvents can be evaporated from compounds of interest to be used for tracking and/or treatment. The human microparticles can be added to the compounds of interest and the mixture agitated for 15 minutes.
The microparticle suspension can then be sonicated for 15 minutes at 15 W of output power to obtain compound intercalation to the microparticles. The resulting particles can be run over Sephadex G50 columns (Sigma) and fractions collected in 0.2 μm-filtered DPBS for subsequent analysis. Incorporation of a compound of interest can be established using appropriate methodology. For example, lipid mediators can be assessed by ELISA where available or LC-MS/MS-based methods. Similarly, incorporation of a tracking agent can be assessed using appropriate methodology. The size of the particles can be characterized using calibration beads of flow-cytometry and/or conventional electron microscopy.
6
Trametinib Liposome for Melanoma Treatment
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Trametinib was purchased from LC Laboratories (Woburn, MA, USA); 1,2-Dioleoyl-sn-glycero-3 phosphocholine (DOPC) was purchased from Cordenpharma (Liestal, Switzerland); PE 18:0/18:0-PEG2000 was obtained from Lipoid (Ludwigshafen, Germany); cholesterol, chloroform, and a Sephadex G50 column were purchased from Sigma-Aldrich (St. Louis, MO, USA); 6-histidine tagged PEGylated (PEG) YSA (6His-PEG-YSA) was obtained from GenScript Corporation (Piscataway, NJ, USA); DOGS-NTA-Ni was obtained from Avanti (Alabaster, AL, USA); Fetal Bovine Serum (FBS) was procured from Atlanta Biologics (Oakwood, GA, USA); Dulbecco’s modified Eagle’s medium (DMEM) and the BCA protein estimation kit were purchased from ThermoFisher Scientific Inc. (Waltham, MA, USA); Penicillin–Streptomycin–Amphotericin B (PSA) was purchased from MP Biomedicals, LLC (Solon, OH, USA); and ALW-II-41-27 was obtained from Cayman Chemical (Ann Arbor, MI, USA). Other chemicals and solvents were of analytical grade. Melanoma cell lines (A375 and SK-MEL-28) were obtained from American Type Culture Collection (Manassas, VA, USA).
7
Identifying FOXO3a Binding on Par-4 Promoter
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Oligonucleotides representing FOXO3a binding site on the Par-4 promoter sequence were synthesized, and the strands were 5′-labeled using the terminal biotin (Integrated DNA Technologies, Coralville, IA, USA). The biotin-labeled sense strand was annealed to its complementary antisense strand and purified over a Sephadex G-50 column (Sigma-Aldrich). The ER alpha site promoter was used as the positive control.9 (link) The prepared nuclear extracts were incubated with Par-4 promoter regions, and oligos bearing FOXO3a-binding sites were utilized.
8
Chloride Flux Assay for nhTMEM16 Activity
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Cl− flux assay was conducted as described previously21 (link),24 (link),52 (link). Liposomes with Ca2+ or EGTA introduced were equilibrated in external buffer with low KCl (1 mM KCl, 300 mM Na-glutamate, 50 mM HEPES, pH 7.4) by spinning through a Sephadex G50 column (Sigma-Aldrich) pre-equilibrated in external buffer. To complete the experiment, 0.2 mL of the flow through from the G50 column was added to 1.8 ml of external solution and the total Cl− content of the liposomes was measured using an Ag:AgCl electrode after disruption of the vesicle by addition of 40 μL of 1.5 M n-octyl-β-D- glucopyranoside (Anatrace). The fraction of liposomes containing at least one active nhTMEM16 ion channel, A, was quantified as follows: where ΔCl is the change in [Cl−] recorded upon detergent addition and ΔClPF is the Cl− content of protein-free liposomes prepared in the same lipid composition on the same day.
9
Quantification of Cl- Flux through nhTMEM16
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Cl− flux assay was conducted as described previously14 . In order to exchange the extracellular ionic composition, liposomes were equilibrated in the external buffer (1 mM KCl, 300 mM Na-glutamate, 50 mM HEPES, pH 7.4) by spinning through a Sephadex G50 column (Sigma-Aldrich) pre-equilibrated in external buffer, 0.2 mL of the flow through was added to1.8 ml of external solution and the total Cl− content of the liposomes was measured using an Ag:AgCl electrode after disruption of the vesicle by addition of 40 μL of 1.5 M n-octyl-β-D-glucopyranoside (Anatrace). The fraction of liposomes containing at least one active nhTMEM16 ion channel, A, was quantified as follows:
where ΔCl is the change in [Cl−] recorded upon detergent addition and ΔClPF is the Cl− content of protein-free liposomes prepared in the presence of 0.5 mM Ca2+ and in the same lipid composition.
where ΔCl is the change in [Cl−] recorded upon detergent addition and ΔClPF is the Cl− content of protein-free liposomes prepared in the presence of 0.5 mM Ca2+ and in the same lipid composition.
10
Radioiodination and Binding of IL-8 to CXCR1
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IL-8 was radioiodinated using the chloramine-T method and purified by chromatography on a Sephadex G-50 column (Sigma-Aldrich) in 0.01 M acetic acid and 0.1% bovine serum albumin (BSA). HEK293 cells were transfected with CXCR1 (300 ng of DNA/well in 12-well plates) with Effectene® (Qiagen, Hilden, Germany). Forty-eight hours later, the cells were washed and incubated for 1 h with binding buffer (serum-free DMEM with 0.1% BSA, pH 7.4) containing 100,000 cpm 125I-labeled IL-8 in the presence of 200 nM of cold ligand. The cells were washed twice with ice-cold PBS. Cell lysates were resolved in 1% SDS and 0.2 M NaOH and radioactivity was determined using a Wallac 1489 Wizard 3 γ-counter (PerkinElmer Inc., Waltham, MA).
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