The direct capture method was conducted using the Maxwell Enviro Wastewater TNA kit (Promega), according to the manufacturer's protocol. A 40 mL seeded sample was added into a 50 mL tube, followed by 500 μL protease solution. The mixture was incubated for 30 min and centrifuged at 3000 ×g for 10 min at room temperature. The supernatant was transferred into two 50 mL tubes to obtain a total volume of ~20 mL for each. Then, 5.5 mL Binding Buffer 1 and 0.5 mL Binding Buffer 2 were added to those two tubes containing the samples to obtain a total volume of ~26 mL each. The tubes were shaken gently, and 24 mL isopropanol (Kanto Chemical) was added to those tubes to obtain a total volume of 50 mL each. The mixtures were shaken gently before passing through the PureYield Midi Binding Column using a vacuum manifold. The PureYield Midi Binding Column was then washed using 5 mL Column Wash 1, followed by 30 mL Column Wash 2. The nucleic acid in the PureYield Midi Binding Column was recovered by 0.5 mL nuclease-free water preheated at 60 °C to obtain a concentrated sample with a final volume of 300 to 400 μL.
Isopropanol
Manufactured by Kanto Chemical
Sourced in Japan
Isopropanol is a clear, colorless, and flammable liquid chemical compound. It is commonly used as a solvent and disinfectant in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Kanto Chemical (3 mentions)
Chloroform, by Kanto Chemical (2 mentions)
Ammonium acetate, by Merck Group (2 mentions)
Acetonitrile, by Kanto Chemical (2 mentions)
Butylated hydroxytoluene bht, by Merck Group (1 mentions)
Silpot 184, by Dow (1 mentions)
Phenyl isothiocyanate, by Fujifilm (1 mentions)
Milli q gradient system, by Merck Group (1 mentions)
Formic acid, by Fujifilm (1 mentions)
Nist srm1950 plasma, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Ceramide sphingoid internal standard mixture 2, by Avanti Polar Lipids (1 mentions)
Sphingosine 1 phosphate (s1p), by Avanti Polar Lipids (1 mentions)
5 protocols using isopropanol
1
Wastewater TNA Extraction Protocol
2
Quantification of Plasmalogens Using LC/MS
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LC/MS grade solvents, including chloroform, methanol, water, and isopropanol, were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). The mobile phase additive ammonium acetate and the antioxidant butylated hydroxytoluene (BHT) were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA). The two plasmalogen internal standards, PlsCho-p16:0/17:0 and PlsEtn-p16:0/17:0, were previously prepared by chemical synthesis in our laboratory [17 (link)]. Other chemicals were of analytical grade and obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless otherwise specified.
3
PDMS Ceiling Chip Fabrication
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The PDMS ceiling (PC) chip was produced by mixing PDMS prepolymer and a curing reagent (Silpot 184; Dow Corning) at an 11:1 weight ratio. The mixture was poured over a mold master, placed in a vacuum chamber for degassing, and moved to an oven to be heated at 72 °C for 1.5 h for curing. After cooling, the solidified PDMS was peeled off from the master. This PDMS disk was diced into individual chips using a cutter knife. For sterilization of the PC chip, they were exposed to UV light for over one hour before use. The mold master was produced as previously reported using conventional photolithography and soft lithography techniques7 (link),40 (link),41 (link).
Briefly, a negative‐type photoresist material (SU‐82100 & 2075; MicroChem Co.) was applied to a 4‐inch wafer via spin coating to achieve uniform thicknesses of 60, 100, and 160 μm. After prebaking process, ultraviolet light was shone through a photomask to delineate a cultivation area, followed by postbaking. The baked mold master was then developed by incubation in propylene glycol monomethylether acetate (GODO, Tokyo, Japan) for 20–30 min, followed by rinsing in isopropanol (code 32435‐70; Kanto Chemical, Kanto, Japan). This SU‐8 mold master can be used repeatedly for the replica molding of PC chips. All mold masters were fabricated by the Micro/Nano Technology Center at Tokai University.
Briefly, a negative‐type photoresist material (SU‐82100 & 2075; MicroChem Co.) was applied to a 4‐inch wafer via spin coating to achieve uniform thicknesses of 60, 100, and 160 μm. After prebaking process, ultraviolet light was shone through a photomask to delineate a cultivation area, followed by postbaking. The baked mold master was then developed by incubation in propylene glycol monomethylether acetate (GODO, Tokyo, Japan) for 20–30 min, followed by rinsing in isopropanol (code 32435‐70; Kanto Chemical, Kanto, Japan). This SU‐8 mold master can be used repeatedly for the replica molding of PC chips. All mold masters were fabricated by the Micro/Nano Technology Center at Tokai University.
4
Lipid Profiling of Human Plasma
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Acetonitrile, chloroform, isopropanol, and methanol were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) for the LC/MS analyses. Formic acid, ammonium formate (1 mol/L) and phenyl isothiocyanate (PITC) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Purified water was obtained from a Milli-Q Gradient system (Millipore, Billerica, MA, USA). Ethanol was purchased from Nacalai Tesque (Kyoto, Japan). The mixture of ISs (UltimateSPLASHTM ONE) and the chemical standards of lipid species were obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA) and Millipore-Sigma Ltd. (Burlington, MA, USA). Pooled normal human plasma, Na EDTA (IPLA-N, Lot: 26393), was purchased from Innovative Research, Inc. (Novi, MI, USA). The reference material (NIST SRM 1950 plasma, NIST QC) [61 (link)] was purchased from Merck Sigma-Aldrich (Darmstadt, Germany).
5
Lipidomics Analysis of Sphingolipids
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The LC/MS grade solvents methanol, acetonitrile, and isopropanol were obtained from Kanto Chemical Co. Ltd (Tokyo, Japan). The mobile phase additives acetic acid, ammonium acetate, and extraction solvent chloroform of LC/MS grade were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ceramide/Sphingoid internal standard mixture II and sphingosine-1-phosphate were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Sphingosine was obtained from Funakoshi Frontiers in Life Science (Tokyo, Japan). Phosphate-buffered saline (PBS) was purchased from Gibco/Life Technologies (Carlsbad, CA, USA).
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