Stability of evolved receptor variants was measured in membrane fractions of transiently transfected HEK293T cells by determining the residual receptor-bound ligand after a heat challenge of the membranes. In the case of PTH1R, the ECD (residues 1–170) was removed from the expression constructs to restrict stability measurements to the TMD. For NTR1, cells were left unmodified, whereas for PTH1R, cells were labeled with 50 nM SNAP-Lumi-4Tb before membranes were prepared as described above. Membranes were then incubated for 2–4 h on ice in ligand-binding buffer containing 20 nM of [3,11-tyrosyl-3,5-3H(N)]-neurotensin (Perkin Elmer) and 500 nM of M-PTH(1–14)-HL647 for NTR1 and for PTH1R, respectively. Where indicated, 25 µM of mini-Gs protein were added to the membrane fractions prior to ligand addition. Thereafter, 0.5 µg of membranes were distributed per well of a 96-well plate and heated to a specific temperature in a PCR thermocycler for 20 min. NTR1-containing membranes were then immobilized on glass fiber filters (Millipore), washed four times with binding buffer, and the residual activity of the radio-ligand was measured on a MicroBeta Plus 1450 liquid scintillation counter (Perkin Elmer). For PTH1R, residual ligand binding was determined by HTRF as described above. Data were analyzed by nonlinear regression fitting.
Glass fiber filters
Manufactured by Merck Group
Sourced in United States
Glass fiber filters are a type of laboratory equipment used for filtration processes. They are composed of fine glass fibers that are bonded together to create a porous, high-surface-area material. Glass fiber filters are designed to effectively capture and retain a wide range of particulates, including small and fine particles, during various laboratory applications.
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Lab products found in correlation
Eutech ph 700 meter, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Optiphase supermix scintillation cocktail, by PerkinElmer (1 mentions)
96 well filter plate, by Merck Group (1 mentions)
Prism 7, by GraphPad (1 mentions)
Organic solvents, by Thermo Fisher Scientific (1 mentions)
3h thymidine, by GE Healthcare (1 mentions)
Tnf α, by Merck Group (1 mentions)
11 protocols using glass fiber filters
1
Measuring Receptor Stability in Membranes
2
Radioligand Binding Assay for α2A-Adrenergic Receptor
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Total radioligand binding was assessed by incubating 5 µg of membrane protein with different concentrations (0.04–12 nM) of the antagonist α2AAR radioligand [3H]RX821002. To define unspecific binding, 20 µM phentolamine was added. Competition binding was performed by incubating 2 µg membrane protein with 0.3–2.0 nM [3H]RX821002 and increasing concentrations of the different α2AAR ligands in the presence (=low affinity state for agonists) and absence (=high-affinity state for agonists) of 10 µM GTP. Following incubation for 1 h at room temperature, membranes were transferred to Millipore glass-fiber filters via vacuum filtration. These filters were incubated with scintillation cocktail and membrane-bound radioactivity was measured with a scintillation counter.
3
Comprehensive Water Quality Assessment
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The air temperature, water temperature and conductivity were measured on-site using a portable EC/TDS TEMP Waterproof Combo Meter (C-100, HM Digital Inc., Culver City, CA, USA). The pH was measured using a portable Eutech pH700 meter (Thermo Fisher Scientific Inc., Waltham, MA, USA). One hundred ml of water sample in triplicate was filtered using glass fiber filters (Millipore, St. Louis, MO, USA); Chlorophyll a (Chl a) was extracted using 90% acetone and measured by spectrophotometry following the method of American Public Health Association (APHA, 2005 ). The samples for nutrient analysis were stored at −20°C soon after arrival to the lab and the parameters such as total nitrogen (TN), total phosphorus (TP), nitrate nitrogen (NO3-N), nitrite nitrogen (NO2-N), ammonium nitrogen (NH4-N), phosphate (PO4-P), and total organic carbon (TOC) were measured as reported in our earlier studies (Vadde et al., 2018 (link); Yuan et al., 2019 (link)). All the values of physico-chemical parameters were compared to the class III water quality standards set by the Chinese Ministry of Environmental Protection (now called as Ministry of Ecology and Environment, People’s Republic of China) (MEP, 2002 ) and correlated with the results of ARGs.
4
Radioligand Binding Assay for MOR Expression
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Human embryonic kidney 293 cells constitutively expressing MOR (HEK-MOR) (Dr. Ping-Yee Law; University of Minnesota Medical School) were harvested and homogenized in membrane preparation buffer (50 mM Tris–HCl at pH 7.4, containing 2 mM ethylenediaminetetraacetic acid [EDTA]) containing a fresh protease inhibitor cocktail (Roche, Basel, Switzerland) and then centrifuged at 30,000g for 30 min. The pellets were resuspended, aliquoted, and stored at − 80 °C. For the [3H]diprenorphine saturation binding assays, membranes (containing 25 μg of protein) were incubated with different concentrations (0.5–5 nM) of [3H]diprenorphine in binding buffer (50 mM Tris–HCl at pH 7.4, containing 2 mM EDTA) at 25 °C for 1 h. For the competitive binding experiments, [3H]diprenorphine (1 nM) was incubated with membranes (containing 25 μg of protein) in the absence or presence of various concentrations of compounds at 25 °C for 1 h. The samples were then rapidly filtered onto glass-fiber filters (Millipore, Billerica, MA, USA) and washed three times with ice-cold phosphate-buffered saline. The radioactivity was quantified using a liquid scintillation counter49 (link).
5
Measuring DNA Synthesis in Response to UV and H2O2
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For experiments using UV irradiation, overnight cultures were diluted 1:100 and grown at 37°C in DGCthy to an OD600 of 0.25–0.35. Where indicated manganese was added to the medium as described above. At this time, half of the cells were mock irradiated, while the other half of the culture was irradiated with 50 J/m2.
For experiments using H2O2, overnight cultures were diluted 1:100 and grown at 37°C to an OD600 of 0.25–0.35 in DGCthy supplemented with 200 µM MnCl2 where indicated. At this time, half of the cells were mock treated, while the remaining culture was exposed to 10 mM H2O2 for 5 min at 37°C. Following either mock or H2O2 treatment, cells were filtered on 0.45-µm membranes to remove excess H2O2 from the medium and resuspended in fresh DGCthy medium either supplemented with Mn or not based on initial growth conditions.
For both UV irradiation and H2O2 experiments, cultures were returned immediately to 37°C after treatment to allow recovery and continued growth. At the times indicated, duplicate 0.5-ml aliquots of culture were pulse-labeled with 0.5 µCi/ml [3H]thymidine for 2 min at 37°C. Cells were then lysed, and the DNA was precipitated in cold 5% trichloroacetic acid and filtered onto Millipore glass fiber filters. The amounts of 3H on each filter were determined by scintillation counting.
For experiments using H2O2, overnight cultures were diluted 1:100 and grown at 37°C to an OD600 of 0.25–0.35 in DGCthy supplemented with 200 µM MnCl2 where indicated. At this time, half of the cells were mock treated, while the remaining culture was exposed to 10 mM H2O2 for 5 min at 37°C. Following either mock or H2O2 treatment, cells were filtered on 0.45-µm membranes to remove excess H2O2 from the medium and resuspended in fresh DGCthy medium either supplemented with Mn or not based on initial growth conditions.
For both UV irradiation and H2O2 experiments, cultures were returned immediately to 37°C after treatment to allow recovery and continued growth. At the times indicated, duplicate 0.5-ml aliquots of culture were pulse-labeled with 0.5 µCi/ml [3H]thymidine for 2 min at 37°C. Cells were then lysed, and the DNA was precipitated in cold 5% trichloroacetic acid and filtered onto Millipore glass fiber filters. The amounts of 3H on each filter were determined by scintillation counting.
6
Radioligand Binding Assay for α-Synuclein Fibrils
7
Particle Flux in the Iquique OMZ
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The R/V Melville C-MORE expedition BiG RAPA (Biogeochemical Gradients: Role in Arranging Planktonic Assemblages) collected samples between Iquique (20.2° S, 70.1° W)
and Rapa Nui (Easter Island; 27.1° S, 109.3° W), Chile during austral spring (November 18-December 14) in 2010 (Table 1). This study describes samples from a water column profile collected at Station 1, which spanned the OMZ structure off the coast of Iquique (20.3° S). At the time of the cruise, the OMZ core in the studied region spanned ~50-530 m in depth. POM was collected from six water depths (0, 30, 40, 65, 200, and 1900 m) extending from the photic zone, across the oxycline, through the OMZ core and below using a rosette equipped with Niskin bottles. Samples of ~200 L were transferred to darkened carboys, filtered onboard through pre-combusted glass fiber filters (0.7 µm, Millipore) using a peristaltic pump, and kept frozen at -20 °C until extraction in the laboratory. No sediment samples were collected during this cruise.
and Rapa Nui (Easter Island; 27.1° S, 109.3° W), Chile during austral spring (November 18-December 14) in 2010 (Table 1). This study describes samples from a water column profile collected at Station 1, which spanned the OMZ structure off the coast of Iquique (20.3° S). At the time of the cruise, the OMZ core in the studied region spanned ~50-530 m in depth. POM was collected from six water depths (0, 30, 40, 65, 200, and 1900 m) extending from the photic zone, across the oxycline, through the OMZ core and below using a rosette equipped with Niskin bottles. Samples of ~200 L were transferred to darkened carboys, filtered onboard through pre-combusted glass fiber filters (0.7 µm, Millipore) using a peristaltic pump, and kept frozen at -20 °C until extraction in the laboratory. No sediment samples were collected during this cruise.
8
Extraction and Analysis of Microplastics from Takeout Containers
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PMFC samples were extracted for MPs, following previous methods (Du et al. 2020 (link); Fadare et al. 2020 (link)). In brief, 150 mL of pure water at 95 °C was transferred to individual PMFCs, covered with their lids, and then shaken at 20 rpm for 30 min. This extraction procedure was repeated once more to simulate the process of delivering takeout food, and then the solvents (i.e., pure water) were transferred to clean glass beakers. After that, PMFC samples were filled with 150 mL of pure water at 30 °C and then covered with their lids. After that, these samples were shaken at 50 rpm for 20 min. This extraction procedure was repeated one more time using the same method, simulating the custom of eating takeout Chinese food (i.e., the interaction between foodstuff and food containers). Finally, all of the solvents obtained in the above extraction processes were combined (total around 600 mL) and then filtrated with glass fiber filters (45 μm pore; Merck-Millipore; Boston, MA, USA). After that, these filters were individually wrapped with aluminum foils, dried in an oven at 40 °C, and then stored at − 20 °C until further analysis.
9
Comparative Analysis of E-Cigarette Constituents
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Infasurf® (lot: 112,809,225) was a generous gift from ONY Inc. (Amherst, NY). All organic solvents used in these studies were purchased from Fisher Scientific (Hampton, NH). Purified water used for all the experiments was obtained from an ELGA PURELAB Classic water purifier (High Wycombe, UK) and was used with a resistivity of 18.2 MΩ·cm. Unflavored, as well as berry- and mint-flavored e-cigarettes (all at 2.4% nicotine content) were purchased from Blu (Charlotte, NC). Conventional research cigarettes (1R6F) were purchased from the University of Kentucky Center for Tobacco Products (Lexington, KY). Glass fiber filters used to capture tar were purchased from EMD Millipore (Billerica, MA). Nicotine, isoprene, and acetaldehyde were all purchased from Fisher Scientific.
10
Quantifying ASMC Proliferation Dynamics
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ASMCs were plated in 24-well cluster plates at a density of 1×105 cells/well in a medium containing 10% FCS and grown to 95% confluence. ASMCs were stimulated with CSE (5 or 15%) or TNF-α (1 ng/ml; Sigma-Aldrich) for 24 h. Then the cells were cultured with [3H]-thymidine (0.25 μCi/ml; GE Healthcare Life Sciences, Chalfont, UK) for 24 h, harvested onto glass-fiber filters (Merck Millipore), and washed twice with PBS and twice with ice-cold 5% trichloroacetic acid (Guangzhou Whiga Technology Co., Ltd.). Subsequently, the cells were dissolved in 0.5 ml NaOH (1 M). Incorporated [3H]-thymidine was quantified by liquid-scintillation counting (Beckman Coulter Inc., Brea, CA, USA).
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