Fasting blood samples were collected at 26-28 weeks’ gestation. Blood lipid extraction was undertaken with chloroform/methanol (Fisher Scientific, Hampton, NH, USA). Plasma phosphatidylcholine (PC) was separated by solid-phase extraction. Fatty acid methyl esters were prepared by reaction with methanolic sulphuric acid and then separated by gas chromatography (BPX-70 column mounted on a Hewlett-Packard HP6890) and detected by flame ionization before quantification as μg/mL of plasma. Inter- and intra-assay variation coefficients for all fatty acids identified in plasma PC were lower than 6% and 3%, respectively. Eleven PUFAs were identified (Supplemental Figure 1 ) and expressed as % of total fatty acids. Additional details have been published previously (28 (link)).
Chloroform methanol
Manufactured by Thermo Fisher Scientific
Sourced in United States
Chloroform/methanol is a laboratory reagent commonly used in the extraction and purification of lipids, nucleic acids, and other biomolecules from biological samples. It is a mixture of chloroform and methanol in a specific ratio that facilitates the separation of these molecules based on their solubility characteristics. The core function of chloroform/methanol is to provide a reliable and efficient method for the isolation and concentration of target analytes from complex matrices, enabling further analysis or downstream processing.
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Lab products found in correlation
Acetonitrile, by Thermo Fisher Scientific (4 mentions)
Hp 6890, by Hewlett-Packard (3 mentions)
Isopropanol, by Thermo Fisher Scientific (2 mentions)
Ammonium formate, by Merck Group (2 mentions)
Beckman lx20 pro analyser, by Beckman Coulter (1 mentions)
Advia 2400 chemistry system, by Siemens (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
S trap micro, by Protifi (1 mentions)
Lys c, by Fujifilm (1 mentions)
Sodium dodecyl sulfate (sds), by Thermo Fisher Scientific (1 mentions)
Phosphoric acid, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Irt peptide, by Biognosys (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Trypsin, by Promega (1 mentions)
Ultra performance liquid chromatography system uplc, by Waters Corporation (1 mentions)
Quadrupole time of flight mass spectrometer q tof ms, by Waters Corporation (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
9 protocols using chloroform methanol
1
Plasma Phosphatidylcholine Fatty Acid Analysis
2
Gestational Diabetes Mellitus and PUFA Profiles
3
Lipid Extraction from Urinary Microvesicles
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Lipids from urinary microvesicles and exosomes were extracted using protocol described previously with slightly modification41 (link). Briefly, 600 µl of 2:1(v/v) chloroform/methanol (Fisher Scientific; Loughborough, UK) was added into 200 µl of suspension containing microvesicles or exosomes (isolated/purified from 300 ml pooled urine) and mixed at 25 °C for 15 min. Separation of organic solution into two phases was performed by adding 100 µl deionized water, mixed for 1 min and centrifuged at 1000 g and 20 °C for 15 min. The organic phase containing lipids (lower layer) was collected using a pipette and transferred into a new tube. The lipid extract was then dried in a SpeedVac concentrator (Savant; Holbrook, NY). The dried lipids were weighed to determine the lipid amount derived from microvesicles or exosomes.
4
Proteome Preparation via S-Trap Digestion
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Samples were solubilized and digested per the S-Trap Micro (Protifi) manufacturer’s protocol2 . Briefly, samples were solubilized in 50 μL of extraction buffer containing 5% sodium dodecyl sulfate (SDS, Affymetrix), 50mM TEAB (pH 8.5, Sigma), and protease inhibitor cocktail (Roche cOmplete, EDTA free), reduced in 5mM TCEP (Thermo), alkylated in 20mM iodoacetamide (Sigma), then acidified with phosphoric acid (Aldrich) to a final concentration of 1.2%. Samples were diluted with 90% methanol (Fisher) in 100 mM TEAB, then loaded onto an S-trap column and washed three times with 50/50 chloroform/methanol (Fisher) followed by three washes of 90% methanol in 100 mM TEAB. A 1:10 ratio (enzyme: protein) of Trypsin (Promega) and LysC (Wako) suspended in 20μL 50mM TEAB was added, and samples were digested for 1.5 hours at 47 °C in a humidity chamber. After incubation, peptides were eluted with an additional 40 μL of 50 mM TEAB, followed by 40 μL of 0.1% trifluoroacetic acid (TFA) (Pierce) in water, and finally 40 μL of 50/50 acetonitrile:water (Fisher) in 0.1% TFA. Eluates were combined and desalted directly using Phoenix peptide cleanup kit (PreOmics) per manufactures protocol, dried by vacuum centrifugation and reconstituted in 0.1% TFA containing iRT peptides (Biognosys, Schlieren, Switzerland).
5
Plasma Phospholipid Fatty Acid Profile
6
Targeted Metabolite Profiling Assay
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For glucose/glutamine consumption assay, cells were cultured in medium for 48 h. One milliliter of culture medium was collected to detect the glucose and glutamine in culture medium. For the detection of other metabolites in cells, approximately 1 × 107 cells were collected for targeted metabolic analysis. The culture medium and cells were then mixed with 0.8 mL of chloroform/methanol (2:1, v/v, Thermo Fisher Scientific) and homogenized for 2 min. The mixture was incubated at -20 °C for 15 min and centrifuged at 13000 g for 10 min at 4 °C. The upper phase was then evaporated under a nitrogen stream. Metabolites were redissolved in a 100 μL solution of acetonitrile/water (99:1, v/v, Anpel laboratory technologies, Shanghai, China) and detected using an Ultra Performance Liquid Chromatography system (UPLC, Waters Corporation, Milford, MA, USA) coupled with a Quadrupole Time-Of-Flight mass spectrometer (Q-TOF MS, Waters Corporation). Standard metabolites for TCA intermediates, glucose, and glutamine were purchased from Sigma-Aldrich. All data were analyzed with the MassLynx software (Waters Corporation) and normalized with cell numbers.
7
Lipid Extraction Reagents Protocol
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Chloroform methanol, acetonitrile, isopropanol, and acetonitrile (≥99.9%) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ammonium formate (≥99.9%) was purchased from Sigma-Aldrich (Shanghai, China). All other reagents commonly used for lipid extraction were obtained from Tedia Company Inc. (Fairfield, OH, USA).
8
Lipid Profiling of Freeze-Stored Shrimps
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Shrimps (L. vannamei) with a height of ~15 cm were purchased from Yonghui supermarket (Fuzhou, China) and transported with ice for 1 h. Upon arriving, all 24 samples were washed under flowing deionized water and packed into 4 bags (6 samples for each bag). Of these 4 bags, 3 were randomly kept under three temperature conditions- 4 °C (refrigerated treatment, RT), −2 °C (particle-freezing treatment, PFT), and −18 °C (frozen treatment, FT) for low-temperature storage (for 10, 30, and 60 days, respectively), while one was treated as a fresh group (RAW) for further analysis.
Ammonium formate was purchased from Sigma-Aldrich (Shanghai, China). Chloroform methanol, acetonitrile, isopropanol, and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other common reagents for lipid extraction were purchased from Tedia Company Inc. (Fairfield, OH, USA).
Ammonium formate was purchased from Sigma-Aldrich (Shanghai, China). Chloroform methanol, acetonitrile, isopropanol, and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other common reagents for lipid extraction were purchased from Tedia Company Inc. (Fairfield, OH, USA).
9
Liver Lipid Extraction and Triglyceride Quantification
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The lipids from 50 mg of frozen liver were extracted and purified according to the method of Folch69 (link). Liver lipids were extracted using chloroform-methanol (2:1, v:v; Thermo Scientific) and an aliquot of the organic phase was collected, dried and resuspended in duplicate in Infinity-TAG lipid-stable reagent (Thermo Scientific). TAG levels were determined according to the manufacturer’s instructions and lipids were quantified using a TAG chemistry calibrator (Pointe Scientific Inc., MI, USA). Serum TAG levels were analysed in duplicate using the commercial L-Type TAG M kit (Wako Diagnostics, Neuss, Germany).
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