WT and mutant P-gps were transiently expressed by PEI-mediated transfection35 (link) in suspension culture-adapted HEK293 cells (FreeStyle 293-F cells; Thermo Fisher). Cells were transfected with a mixture of PEI-MAX (Mw 40,000; Polysciences) and plasmids at final concentrations of 4 μg ml−1 and 1 μg ml−1, respectively. For purification, P-gp-expressing cells were solubilized with 1% C12E8 in solubilization buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-Na (pH 7.2), 150 mM NaCl, 50 mM KCl, 10% glycerol, and 1 mM 2-mercaptoethanol) supplemented with protease inhibitors (Complete EDTA-free; Roche Applied Science). After insoluble materials were removed by centrifugation (45,000 × g, 30 min), ANTI-FLAG M2 Affinity Gel (A2220, Sigma-Aldrich) was added, and the sample was incubated for 2 h. Proteins were eluted in solubilization buffer containing 0.05% C12E8 and 0.15 mg ml−1 each of FLAG peptide and 3× FLAG peptide, and then concentrated to 0.5−2 mg ml−1 using Amicon Ultra 0.5 ml filters (100,000, Merck Millipore).
Amicon ultra 0.5 ml filter
Manufactured by Merck Group
Sourced in United States, Germany
The Amicon Ultra 0.5 ml filters are a type of centrifugal filter device used for the concentration and purification of macromolecules such as proteins, enzymes, and nucleic acids. The filters feature a selectively permeable membrane that allows the passage of small molecules while retaining the larger target molecules. They are designed for rapid sample preparation and are commonly used in research and diagnostic laboratory settings.
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Lab products found in correlation
Lsm 880, by Zeiss (2 mentions)
Glutathione sepharose 4b bead column, by GE Healthcare (2 mentions)
Proteiniso ni nta resin, by Transgene (2 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
Forskolin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Complete edta free, by Roche (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
Griess reagent kit, by Dojindo Laboratories (1 mentions)
No2 no3 assay kit c 2, by Dojindo Laboratories (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
His select nickel affinity gel, by Merck Group (1 mentions)
Mounting solution, by Abcam (1 mentions)
Dulbecco s modified eagle medium dmem, by Welgene (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Airyscan, by Zeiss (1 mentions)
27 protocols using amicon ultra 0.5 ml filter
1
Transient Expression and Purification of P-glycoprotein
2
Isolation of Extracellular Vesicles from Aspergillus-Infected Cells
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After infection with A. fumigatus , the supernatant of dPLB cells was collected, and extracellular vesicles were isolated using two different methods: a differential centrifugation-based approach or a size exclusion chromatography-based approach. The first method was described previously for the isolation of neutrophil-derived extracellular vesicles (10 (link), 33 (link)). In both approaches, samples were centrifuged at 3,000 × g for 15 min at 4°C and filtered through 5-μm-pore-size filters (Carl Roth). In the first method, samples were then centrifuged at 19,500 × g for 20 min at 4°C to collect extracellular vesicles. For the second approach, the clarified filtrate was concentrated using Amicon Ultra-15 centrifugal filters (Merck) with a molecular mass cutoff (MWCO) of 100 kDa for 10 min at 4°C and 3,220 × g and loaded on size exclusion chromatography qEV 70-nm columns (Izon). After the 3-ml void volume was discarded, 1.5 ml of extracellular vesicle sample was collected and measured. When necessary, extracellular vesicles were further concentrated using 10-kDa cutoff Amicon Ultra 0.5-ml filters (Merck). Extracellular vesicles were used fresh for most of the downstream experiments except for proteomics analysis where they were frozen at −20°C.
3
Nitric Oxide Quantification in Fungal Cells
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Fungal cells were grown in GMM(Pro) medium, and the stress-treated cell extracts were obtained using the same method described above. After quantifying the amounts of the proteins, the proteins were removed from the cell extracts using Amicon Ultra 0.5-mL filters (Merck, Darmstadt, Germany). The resultant samples were transferred to a 96-well plate, and the NO level was quantified using an NO2/NO3 Assay Kit-C II(Colorimetric) ~ Griess Reagent Kit ~ (Dojindo) at 540 nm.
4
Recombinant Protein Expression and Purification
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The protein expression constructs were transformed into the BL21 (DE3) Rosetta strain (Merck Millipore). The selected bacterial cells carrying the constructs were inoculated into 30 mL liquid Luria-Bertani (LB) media containing the kanamycin sulphate antibiotic (30 μm/mL), 2% (v/v) glucose, and were grown overnight at 37 °C. The following day, cells were diluted to 200 mL with liquid LB media containing the same antibiotics, 0.4% (v/v) glucose, and grown until OD600 reached the 0.6 value. Protein expression was induced using 1 mM Isopropyl β- d-1-thiogalactopyranoside (IPTG; Thermo Fisher Scientific); then, the cells were incubated at 37 °C for 2 h with constant shaking. Expressed proteins were then purified with the HIS-Select nickel affinity gel (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions. Purified proteins were concentrated using Amicon Ultra 0.5 mL filters (Merck Millipore); and then, stored in 40% (v/v) glycerol at −20 °C for later use.
5
Uptake of Exosomes in Cell Lines
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Dulbecco's Modified Eagle Medium (DMEM) was purchased from Welgene (South Korea). Fetal Bovine Serum (FBS) was purchased from Gibco (USA). All other materials were purchased from Sigma-Aldrich. RAW264.7 (ATCC, number TIB-71) and IEC-18 (Korean Cell Line Bank, number 21589) cells were cultured in DMEM supplemented 10% (v/v) FBS, and incubated at 37 °C in 5% CO2.
Mi-Exo was labeled with 2.5 μM DiO (Life Technologies, USA) and incubated at 37 °C for 20 min, and then concentrated at 14 000 × g for 20 min at 4 °C using a 30k molecular weight cut-off (MWCO) centrifugal filter (Amicon® Ultra 0.5 mL Filters, Merck Millipore, USA).
Cell uptake assay was performed as below. The cells (RAW264.7 and IEC-18 cells, 3 × 104 cells per well) were placed onto a 10 mm cover slip in a 24-well plate, followed by incubation for 2 days. After the washing process, DiO-labeled Mi-Exo was added, and the cells were further cultured for 24 h (at 37 °C in 5% CO2). The cells were fixed with 4% chilled paraformaldehyde for 30 min, and washed 3 times in DPBS. Next, they were stained with DAPI for 5 min, and washed 3 times again. Mounting solution (Abcam, UK) was added to the cell-coated cover slip part and dried for O/N. The cell fixation step was performed at RT. The cell uptake image was photographed using a model LSM-880 with Airyscan and super-resolution confocal laser scanning microscopy (ZEISS, Germany).
Mi-Exo was labeled with 2.5 μM DiO (Life Technologies, USA) and incubated at 37 °C for 20 min, and then concentrated at 14 000 × g for 20 min at 4 °C using a 30k molecular weight cut-off (MWCO) centrifugal filter (Amicon® Ultra 0.5 mL Filters, Merck Millipore, USA).
Cell uptake assay was performed as below. The cells (RAW264.7 and IEC-18 cells, 3 × 104 cells per well) were placed onto a 10 mm cover slip in a 24-well plate, followed by incubation for 2 days. After the washing process, DiO-labeled Mi-Exo was added, and the cells were further cultured for 24 h (at 37 °C in 5% CO2). The cells were fixed with 4% chilled paraformaldehyde for 30 min, and washed 3 times in DPBS. Next, they were stained with DAPI for 5 min, and washed 3 times again. Mounting solution (Abcam, UK) was added to the cell-coated cover slip part and dried for O/N. The cell fixation step was performed at RT. The cell uptake image was photographed using a model LSM-880 with Airyscan and super-resolution confocal laser scanning microscopy (ZEISS, Germany).
6
Plasma Amino Acid and Gut Hormone Analysis
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Blood samples were centrifuged (3,000 r/min) at 4 °C for 10 min within 1 h from collection and the plasma aliquoted into Eppendorf tubes before frozen at −80 °C until AA analysis. To prepare the plasma for the AA evaluation, samples were first centrifuged at 14,000 × g at room temperature for 30 min using Amicon® Ultra 0.5 mL filters (3 kDa MWCO) (Merck Millipore, Burlington, USA). The filtrate was then aliquoted into Eppendorf tubes and mixed, in the same proportion (1:1), with an internal standard solution containing 2-aminobutanoic acid and sarcosine. AA within the processed samples were then derivatised following the methodology described by Valgepea et al. [18 (link)], and the concentration analysed by using RP-HPLC. Supernatant samples were analysed for total CCK and GLP-1 using a Porcine Cholecystokinin ELISA kit from MyBioSource (MBS264395, San Diego, USA) and a glucagon-like peptide-1 (Total) ELISA kit from Merck Millipore (EZGLPT1-36 K, Burlington, USA), respectively. Intra- and inter-assay coefficients of variation for the CCK kit were 6.0% and 9.3%, respectively, whereas GLP-1 intra- and inter-assay coefficients of variation for the GLP-1 kit were 1.9% and 2.2%, respectively. Optical density recordings of the ELISA plates were performed in BMG LABTECH FLUOstar OPTIMA (BMG Labtech, Mornington, Australia).
7
Purification and Storage of Enzymes
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All enzymes with the exception of Alg2 were purified. For purification, cells were lysed by high-pressure homogenization (three cycles, 1,000 bar, 4°C) followed by centrifugation (7,192 x g, 20 min, 4°C). Supernatants were then applied to an equilibrated immobilized metal affinity chromatography (IMAC) column (Ni Sepharose HP column; Cytiva, Chicago, United States). Purified enzyme solutions were desalted using Amicon® Ultra 0.5 mL filters from Merck (Darmstadt, Germany) with a molecular weight cut-off of 10 kDa according to the manufacturer’s instruction. Desalted enzymes were stored in 50% (v/v) glycerol at −20°C.
8
UCP-LF Strip Test for Quantifying CAA
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The index test is the UCP-LF strip test detecting and quantifying CAA levels. LF strips can be analysed with POC care readers [47 (link)] or, as used in this study, with a multistrip benchtop reader (Upcon; Labrox Oy, Turku, Finland) capable of analysing 20 strips at a time. The UCP-LF CAA test was developed at Leiden University Medical Center (Leiden, The Netherlands) [38 (link), 48 (link)]. So far, the tests are produced in-house at LUMC in a batch-wise manner (ca. 2000 tests per batch), fully quality-controlled using standardized reference samples, ensuring specificity and sensitivity. A dry reagent format is available that can be stored and transported at ambient temperature [39 (link)]. Here, 417 μl of fresh or stored (− 20 °C) urine will be tested by the UCP-LF CAA urine test following published procedures [48 (link)] including a concentration step (centrifugal Amicon Ultra 0.5 mL filters with a 10 kD cut-off; Merck Millipore) after pre-treatment of the urine with 1/6 volume of 12% trichloroacetic acid and (UCAAhT417 format) [49 ]. All measurements will include a set of CAA-spiked standards into a negative urine to determine the threshold of positivity that is set to ≥2 pg/ml.
9
Stability of RAGE-targeted Dendrimer
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The stability of multimodal RAGE-targeted dendrimer was measured in plasma for 24 h post radiolabeling with 64Cu. 64Cu-Rho-G4-CML (~5 mCi) was added to 10 mL of plasma kept at 37 °C with stirring. At 0.5, 1, 1.5, 2, 8, 12, and 24 h post radiolabeling, 0.5 mL of plasma sample was collected and centrifuged using 3k MWCO Amicon Ultra-0.5 mL filters (Millipore, USA). The 64Cu radioactivity (counts-per-minute, CPM) and rhodamine fluorescence (at 627 nm) of both filtrate (unbound 64Cu or rhodamine) and recovered liquid (64Cu-Rho-G4-CML) were measured using a gamma well counter (Wizard2, Perkin-Elmer, USA) and fluorescence microplate reader (BioTek, USA), respectively. Stability expressed as the percent of bound 64Cu or rhodamine (% bound) was calculated by dividing radioactivity or fluorescence of recovered liquid by the total radioactivity or fluorescence of the sample. All stability experiments were done in triplicates.
10
GST Pull-Down Assay Protocol
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GST pull-down assays were conducted as previously described with slight modifications [39 (link)]. Briefly, the encoded GST- or His-tagged fusion proteins and the control GST proteins were expressed in BL21 cells after induction with 0.1 mmol/L IPTG overnight at 18°C. Centrifuged cells were resuspended in lysis buffer (1 × PBS, 0.2 mM PMSF, 1% Triton X-100) and sonicated for 15 min. After centrifugation, the supernatant was applied to a Glutathione–Sepharose 4B bead column (GE Healthcare) or ProteinIso Ni-NTA Resin (TransGen Biotech, China), in accordance with the manufacturers’ instructions. Purified GST/His-tagged fusion proteins were diluted with 1× PBS and filtered through Amicon Ultra 0.5 ml filters (Millipore). Then, 1 μg of purified GST protein or GST fusion protein was captured by the Glutathione–Sepharose 4B beads (GE Healthcare), and His-tagged fusion protein was added for incubation overnight at 4°C. The beads were then washed three times with ice-cold PBS. The supernatant was loaded onto gels, followed by immunoblotting analysis.
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