MeCN (200 μL) was added to the urine samples (50 μL) and vortexed for 30 s. The samples were centrifuged at 12,000 × g for 5 min, and then the supernatants were filtered by Cosmospin Filter G (0.2 μm, Nacalai Tesque, Kyoto, Japan). The filtrates were moved into LC vials and analyzed by HILIC–MS/MS. We also investigated ultrafiltration (Amicon Ultra-0.5, 3 kDa, Merck Millipore, Burlington, MA) instead of microfiltration.
Cosmospin filter g
Manufactured by Nacalai Tesque
Sourced in Japan
The Cosmospin Filter G is a laboratory equipment designed for filtration. It is used to separate solid particles from liquids through the process of centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Ni nta agarose, by Qiagen (2 mentions)
Expi293 expression system, by Thermo Fisher Scientific (2 mentions)
Cev 3100, by Eyela (2 mentions)
Beh shield rp18 column, by Waters Corporation (2 mentions)
Amicon ultracell, by Merck Group (1 mentions)
Ygb05, by Yasui Kikai (1 mentions)
Mb1001c s, by Yasui Kikai (1 mentions)
Sb c18 column, by Agilent Technologies (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Isoproterenol, by Nacalai Tesque (1 mentions)
5 hiaa, by Merck Group (1 mentions)
Agilent masshunter quantitative analysis software, by Agilent Technologies (1 mentions)
Lc 10a, by Shimadzu (1 mentions)
Spd 10a, by Shimadzu (1 mentions)
Cosmosil 5c18 paq column, by Nacalai Tesque (1 mentions)
96 well plate, by Corning (1 mentions)
Crystal screen ht, by Hampton Research (1 mentions)
Index ht, by Hampton Research (1 mentions)
9 protocols using cosmospin filter g
1
Urine Sample Preparation for HILIC-MS/MS
2
Brain Homogenization and LC-MS/MS Analysis
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The dissected whole brain was homogenized in 100 µl of the analytical solution supplemented with 20 pg/µl of isoproterenol using micropestle for 60 s on ice. The homogenate was centrifuged at 1300G at 4 °C for 15 min and the supernatant was transferred to a filtration column (Cosmospin Filter G; hydrophilic PTFE membrane filter with 0.2 μm pores, Nacalai Tesque) and then centrifuged at 3000G at 4 °C for 30 min. Five microliters of the filtered supernatant was subjected for the LC–MS/MS analysis.
3
Recombinant SARS-CoV-2 Spike Protein Production
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SARS-CoV-2 monomeric spike (S) proteins were produced using a mammalian cell protein expression system. The S gene sequence (GenBank: MN908947) was commercially synthesized (Genewiz, Japan). The extracellular region of the S sequence (amino acids 1–1213; MFVF…IKWP) was codon optimized for mammalian cell expression and the polybasic cleavage site was removed (RRAR to A) with stabilizing mutations (K986P and V987P; wild-type numbering) added as described by Amanat et al. [18 (link)]. Expression plasmid of monomeric S comprise the extracellular domain of S that is C-terminally fused to the thrombin site, fibritin trimerization sequence, and a Strep-tag II plus a His tag cloned into a PCXSN vector. S proteins were expressed using the Expi293 Expression System (Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer’s instructions. Seven days post-transfection, the medium was clarified by centrifugation at 1200 × g, filtered, and purified with Ni–NTA Agarose (QIAGEN, Germantown, MD, USA). The purified S proteins were concentrated using Amicon Ultracell (Merck) centrifugation units with a cut-off of 50 kDa, and the buffer was changed to PBS (pH 7.4). The proteins were filtered through Cosmospin filter G with a pore size of 0.2 micron (Nacalai Tesque, Inc.) and stored at − 80 °C until use.
4
Pigment Extraction and Quantification
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Lyophilized cells (2–3 mg) were suspended in 500 μL of methanol:acetone (5:5 [v/v]) and fractured using 0.5 mm glass beads in a multi-bead shocker MB1001C(S) as described for lipid analysis. The samples were centrifuged at 10,000×g for 2 min at 4 °C, and the supernatant was transferred to a new microtube. The extraction procedure was repeated four times to obtain 2 mL of supernatant. The supernatant (330 μL) was dried in a vacuum using an evaporator CEV-3100 (EYELA, Tokyo, Japan), resuspended in 500 μL of chloroform:acetonitrile (2:8 [v/v]) containing 1 μM trans-β-apo-8-carotenal as an internal standard, and filtered using a 0.22 µm Cosmospin Filter G (Nacalai Tesque). Pigments were identified and quantified using an ACQUITY ultra liquid chromatography (UPLC) system equipped with a photodiode array detector and a BEH Shield RP18 column (1.7 μm, 2.1 mm × 100 mm; Waters, Milford, MA, USA) [25 (link), 47 (link)].
5
Quantifying Pigments in Lyophilized Cells
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Lyophilized cells prepared above were used for measurement of carotenoids and chlorophylls. 3 mg of lyophilized cells was suspended in 500 μL of pre-cooled (4 °C) methanol:acetone = 1:1 (v/v), and fractured with 0.5 mm glass beads YGB05 and a multi-bead shocker MB1001C(S) (Yasui Kikai) at 4 °C. After centrifugation at 10,000×g for 5 min, 150 μL of the supernatant was dried under vacuum using a centrifugal evaporator CEV-3100 (EYELA, Tokyo, Japan). The dried sample was resuspended in 500 μL of acetonitrile:chloroform = 8:2 (v/v) containing 1 μM trans-β-apo-8′-carotenal as an internal standard, and filtered by a 0.22 µm Cosmospin Filter G (Nacalai Tesque, Kyoto, Japan). Then samples were subjected to identification and quantification of pigments with an ACQUITY ultra performance liquid chromatography system equipped with a photodiode array detector and a BEH Shield RP18 column (1.7 μm, 2.1 mm × 100 mm; Waters, MA, USA)57 (link).
6
Quantifying 5-HT and 5-HIAA in Tilapia Brain
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5-HT and 5-HIAA measurement in male tilapia brain area 2 and 3 (control = 8, defeat = 8) was carried out using liquid chromatography-mass spectrometry (the Agilent Technologies 6410 Triple Quad Liquid chromatography-mass spectrometry equipped with a ZORBAX SB- C18 column). The procedures were as described previously (4 (link)). Briefly, the brain tissues were homogenized with 350 μL of 20 ng/mL isoproterenol (internal standard, Nacalai Tesque) in 50% acetonitrile (Fisher Scientific) with 0.1% formic acid (Sigma). The supernatant was filtered by Cosmospin filter G (Nacalai Tesque). A standard solution of 5-HT, 5-HIAA (Sigma), 5-HIAA, and 20 ng/mL isoproterenol was prepared in 50% acetonitrile with 0.1% formic acid in the range of 1.25 to 160 ng/mL. The production of 5-HT, 5-HIAA, and isoproterenol was observed for further analysis (m/z 177.1–160 for 5-HT, m/z 192.07–146 for 5-HIAA, and m/z 212.1–194 for isoproterenol). Data acquisition software Agilent Masshunter Quantitative Analysis software (RRID: SCR_015040, Agilent Technologies) was used for data analysis.
7
SARS-CoV-2 Spike Protein Production and Purification
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SARS-CoV-2 monomeric S-ect was produced using a mammalian cell protein expression system. The S gene sequence (GenBank, MN908947) was commercially synthesized (Genewiz). The extracellular region of the S sequence (amino acids 1 to 1213; MFVF…IKWP) was codon-optimized for mammalian cell expression, and the polybasic cleavage site was removed (RRAR to A) with stabilizing mutations (K986P and V987P; wild-type numbering) introduced as described by Amanat et al. (53 (link)). The plasmid expressing monomeric S comprised the extracellular domain of S, which was C-terminally fused to the thrombin site, and a Strep-tag II and a His tag was cloned into the PCXSN vector. S-ect was expressed using the Expi293 Expression System (Thermo Fisher Scientific), according to the manufacturer’s instructions. Seven days after transfection, the medium was clarified by centrifugation at 1200g, filtered, and purified with Ni-NTA agarose (QIAGEN). The purified S-ect was concentrated using Amicon Ultracel (Merck) centrifugation units with a cutoff of 50 kDa, and the buffer was changed to PBS (pH 7.4). Proteins were filtered through a Cosmospin filter G with a pore size of 0.2 μm (Nacalai Tesque) and stored at −80°C until use.
8
High-Performance Liquid Chromatography Analysis
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Aliquots (0.94 ml) of the culture solution were mixed with 10 µl of 0.5 mol/L potassium phosphate buffer (pH 6.8) and 50 µl of methanol, and filtered using the Cosmospin Filter G (0.2 µm; Nacalai tesque, Kyoto, Japan). A 20‐µl aliquot of the sample was injected into an HPLC system (LC‐10AS, Shimadzu, Kyoto, Japan) equipped with a COSMOSIL 5C18‐PAQ column (5 µm, 4.6 mm I.D. × 250 mm; Nacalai tesque, Kyoto, Japan). The eluent consisted of 5 mmol/L potassium phosphate buffer (pH 6.8) and 5% (v/v) methanol, and the flow rate was 0.2 ml/min. The absorbance of the eluate was monitored at 210 nm using a UV detector (SPD‐10A, Shimadzu, Kyoto, Japan).
9
Crystallization of CLA-ER with FMN and KetoC
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For the crystallization of CLA-ER with FMN, 4 mM NADH and 2 mM FMN were added to the protein solution. Initial crystallization trials were performed by the sitting-drop vapor-diffusion method in 96-well plates from Corning (Tokyo, Japan) using the sparse-matrix screening kits, Crystal Screen HT and Index HT from Hampton Research (Aliso Viejo, CA, USA), at 293 K. Drops containing equal volumes (0.2 lL) of protein solution and reservoir solution were equilibrated against a 40 lL reservoir solution. optimization of the crystallization conditions, the crystals of holo CLA-ER with FMN grew under the condition of 0.1 M BisTris (pH 6.0) and 21% poly(ethylene glycol) monomethyl ether 5000.
For crystallization of CLA-ER with both FMN and KetoC, 4 mM NADH, 2 mM FMN and 2 mM KetoC were added to the protein solution and the mixture was incubated at 4 °C overnight. The mixture was filtered with Cosmospin filter G (Nacalai Tesque, Kyoto, Japan) with a pore size of 0.2 lm for crystallization using the same methods as above. After optimization, the crystal of CLA-ER in complex with FMN and KetoC grew under the conditions of 100 mM sodium cacodylate (pH 6.8), 200 mM NaCl and 2.0 M ammonium sulfate.
For crystallization of CLA-ER with both FMN and KetoC, 4 mM NADH, 2 mM FMN and 2 mM KetoC were added to the protein solution and the mixture was incubated at 4 °C overnight. The mixture was filtered with Cosmospin filter G (Nacalai Tesque, Kyoto, Japan) with a pore size of 0.2 lm for crystallization using the same methods as above. After optimization, the crystal of CLA-ER in complex with FMN and KetoC grew under the conditions of 100 mM sodium cacodylate (pH 6.8), 200 mM NaCl and 2.0 M ammonium sulfate.
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