Polycaprolactone (PCL, Mw 80,000 Da, Sigma-Aldrich, Darmstadt, Germany), chitosan (CS, Mw of 50,000–190,000 Da, Sigma-Aldrich), hydroxyapatite (Sigma-Aldrich), chloroform (Merck, Burlington, MA, USA), and acetic acid (AA, 100%, Merck) were purchased from the local supplier (TemadKala Co., Tehran, Iran). All the reagents and the materials were of analytical grade.
Chloroform
Manufactured by Merck Group
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About the product
Chloroform is a colorless, volatile liquid with a characteristic sweet odor. It is a commonly used solvent in a variety of laboratory applications, including extraction, purification, and sample preparation processes. Chloroform has a high density and is immiscible with water, making it a useful solvent for a range of organic compounds.
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Check compatibility
6 372 protocols using chloroform
1
Fabrication of PCL-Chitosan Biocomposite
2
Primary Neuronal Cell Culture Protocol
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Minimum Essential Medium (MEM), Horse serum (HS), B27 supplement, L-glutamine (L-Gln), Penicillin-streptomycin (P/S), Lipofectamine 2000 Transfection Reagent, TRIzol, MultiScribe™ Reverse Transcriptase (RTase), and Antibiotic-Antimycotic (AA) were purchased from Invitrogen (Carlsbad, CA, USA); Neurobasal medium, Sodium bicarbonate, Sodium Pyruvate, Hank’s Balanced Salt Solution (HBSS), Tryptone, Yeast extract, and Opti-MEM were purchased from Gibco (Grand Island, NY, USA); Poly-L-lysine (PLL), Papain, Cysteine, Calcium chloride (CaCl2), Deoxyribonuclease I (DNase I), Glutamate (Glu), Cytosine-β-D-arabinofuranoside (AraC), Sodium chloride (NaCl), Adenosine triphosphate (ATP), Magnesium chloride (MgCl2), Chloroform, EGTA, Formaldehyde solution, Sodium butyrate, IGEPAL CA-630, Dithiothreitol (DTT), Hydrochloric acid (HCl), Phenol solution, sodium deoxycholate, Sodium orthovanadate (SV), and kanamycin sulfate were purchased from Sigma-Aldrich (Saint Louis, MO, USA); Triton X-100, EDTA, Sodium dodecyl sulfate (SDS), Sodium acetate, and Phenylmethyl sulfonyl fluoride (PMSF) were purchased from USB (Santa Clara, CA, USA); Fetal bovine serum (FBS) was purchased from PEAK SERUM (Wellington, CO, USA); SYBR Green PCR master mix and High-capacity cDNA reverse transcription kit were purchased from Applied Biosystems (Waltham, MA, USA); Wizard® Genomic DNA Purification Kit was purchased from Promega (Madison, WI, USA); NEBuffer 2.1, NEBuffer 3.1, CutSmart buffer, Nde1, Nhe1-HF, AgeI, Bglll, HindIII, and T4 ligase were purchased from New England Biolabs (Ipswich, MA, USA); Agarose, proteinase K and Glycine were purchased from Chumeia (Paso Robles, CA, USA); NucleoSpin Gel and PCR Clean-up was purchased from MACHEREY-NAGEL (Düren, Köln, Germany); Tris-HCl, and isopropanol were purchased from Avantor (Radnor, PA, USA); Taq DNA polymerase was purchased from GeneTeks Biosciences (Xinbei, Taiwan); 2.5 mM dNTP was purchased from Genezyme biotech (Miaoli, Taiwan); 100 mM dNTP was purchased from GeneDireX (Taoyuan, Taiwan); plasmid miniprep kit was purchased from Biokit (Miaoli, Taiwan); American Bacteriological Agar was purchased from condalab (Torrejón de Ardoz, Madrid, Spain); RNase A, DNase and protease-free was purchased from Thermo Scientific (Waltham, MA, USA); glycogen was purchased from Roche (Basel, CH); anti-H3K4me1 (polyclonal) (Cat# ab8895), anti-H3K4me1 (monoclonal) (Cat# ab176877), and rabbit polyclonal IgG (Cat# ab37415) were purchased from Abcam (Cambridge, UK). Anti-α-tubulin (GTX112141) and anti-green fluorescent protein (GFP, GTX113617) were purchased from GeneTex (Trvine, CA, USA). IRDye 680RD goat anti-mouse IgG secondary antibody and IRDye 800CW goat anti-rabbit IgG secondary antibody were purchased from LI-COR Bioscience (Lincoln, NE, USA). The BCA protein assay kit was purchased from Merck (Kenilworth, NJ, USA).
3
Phospholipid Extraction from Egg Yolk
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In this study, we used a protocol for isolating phospholipids from egg yolk, which was developed by Gladkowski et al. in 2012. In the protocol, the egg yolk served as the primary raw material for their research experiment. The procedure began with breaking the egg and weighing the yolk. The yolk was then mixed with acetone (Merck, Darmstadt, Germany) and continuously stirred using a magnetic stirrer (IKA, North Carolina, Germany) for 10 min. The mixture was transferred to a glass flask and centrifuged (Eppendorf, Hamburg, Germany) at 3000 rpm for 10 min. After separation and drying of the precipitate, a mixture of chloroform (Sigma-Aldrich, St. Louis, MO, USA) and ethanol (Merck, Darmstadt, Germany) in a ratio of 2:1 was added, and the mixture was stirred thoroughly for 3 h. The resulting solution was then filtered, and the organic solvent was separated to form a thin layer of lipids. The yield of phospholipids extracted from egg yolk is typically calculated as a percentage of the weight of phospholipids obtained relative to the initial weight of the egg yolk used. The formula for calculating the yield is as follows:
4
HPLC Analysis of Phenolic Compounds
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HPLC standards (chlorogenic acid and (−)-epicatechin) and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMSO and organic solvents (chloroform, isopropanol, methanol, and ethanol) were obtained from Sigma-Aldrich, Samchun Chemical (Seoul, Republic of Korea), and Daejung Chemical (Siheung, Republic of Korea). Inorganic salts were from Sigma-Aldrich, and silica gel was from Merck (Rahway, NJ, USA). DMEM and FBS were supplied by Hyclone (Logan, UT, USA), and Antibiotic-Antimycotic was from Gibco (Grand Island, NY, USA).
5
Synthesis and Characterization of 3-MBL Liposomes
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Synthetic N-3-methoxybenzyl-linoleamide (3-MBL) was synthesized in the Organic Chemistry Laboratory at MCPHS University [30 (link)]. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), MPEG(2000)-Distearoyl phosphatidylethanolamine (ammonium salt) (DSPE-PEG2000-OCH3), and CarboxyPEG(5000)-Distearoyl phosphatidylethanolamine (ammonium salt) (DSPE-PEG5000-COOH) were supplied by Nanocs (Natick, MA, USA). N-(3-dimethylaminopropyl)-N’ethyl-carbodiimide hydrochloride (EDC), 2-(N-morpholino) ethanesulfonic acid (MES), N-hydroxysulfosuccinimide (sulfo-NHS), glycine, the Phospholipid Assay kit, and the QuantiPro™BCA Assay kit were obtained from Sigma Aldrich (St. Louis, MO, USA). OX26 and the Pierce™F(ab′)2 Micro Preparation kit were purchased from Fisher Thermo Scientific (Waltham, MA, USA). Pilocarpine, methyl-scopolamine, diazepam, carbamazepine, tetraglycol, and polyethyleneglycol 600 were also obtained from Sigma Aldrich (St. Louis, MO, USA). Chloroform, tert-Butyl methyl ether (t-BME), HPLC-grade methanol (MeOH), and acetonitrile (ACN) were purchased from Merck (Arequipa, Peru). Ultrapure water was freshly obtained before use from a Merck Simplicity water purification system. All the other reagents were purchased from Sigma Aldrich (Arequipa, Peru).
6
Comprehensive RNA Extraction and Analysis
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We extracted total RNA using TRIzol (Roche, Germany) by applying a modified version of the manufacturer’s protocol. Initially, chilled embryos were equilibrated with 250 μL of TRIzol reagent (Omics Bio, Taiwan; Roche, Germany) at room temperature for 10 min. After the embryos were cut and fragmented, they were mixed with 50 μL of chloroform (Sigma, USA) at room temperature for 10 min. The mixture was then centrifuged at 4 °C for 15 min at 12 000 × g. The resulting supernatant was carefully transferred to a sterile Eppendorf tube. The TRIzol and chloroform treatments were repeated. Next, 500 μL of isopropanol (MERCK, Germany) was added and mixed thoroughly with a pipette for 1 min. The mixture was centrifuged at 4 °C for 15 min at 12 000 × g after 10 min. The supernatant was removed after centrifugation, and 1 mL of 75% ethanol (Nihon Shiyaku, Japan) was added to generate a white precipitate. Centrifuging at 4 °C for 5 min (12 000 g) was performed to disperse the precipitate. To remove phenol, washing was performed three times. The resulting ethanol residue was removed with a centrifugal evaporator (CentriVap; Labconco, USA). Finally, 16–20 μL of DEPC-treated distilled water was added to dissolve the RNA pellet. An SSP-3000 nanodrop spectrophotometer (Infinigen Biotech, USA) was used to assess RNA quality and concentration. UniRegion Biotech gel electrophoresis on 1% agarose in tris–acetate-EDTA (TAE) buffer was performed to assess and verify RNA integrity. RNA quality was validated by measuring the absorbance ratio at 260/280, which was determined to be 1.8–2.0. For post-quantification, 1 μg of total RNA was added to each reverse transcription process by using Takara Biosystems’ TaKaRa Primescript Master Mix. To obtain a 10 μL volume, the RNA template was mixed with 2 μL of MasterMix and PCR-grade water in the correct proportion. The PCR protocol is as follows. After the temperature was maintained at 37 °C for 30 min, it was increased to 85 °C for 5 s and subsequently maintained at 4 °C. Quantitative real-time PCR studies were performed using cDNA stored at − 20 °C.
7
Comprehensive Metabolomic Analysis Protocol
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Luria-Bertani broth was obtained from Cyrusbioscience, Inc. (New Taipei City, Taiwan). M9 minimal medium was sourced from Becton, Dickinson and Company (East Rutherford, USA). Casein hydrolysate, FeCl2, aspartic acid, sodium nitrate, 13C-CO2, chloroform, methoxymation hydrochloride, N-tert-Butyldimethylsily-N-methyltrifluoroacetamide (MTBSTFA), pentafluorobenzyl bromide (PFBBr), and methanolic sodium hydroxide were procured from Sigma-Aldrich (Merck Group, St. Louis, USA). Dimethyl sulfoxide (DMSO), thiamine and NiCl2 were procured from Acros Organics (Thermo Fisher Scientific Inc., Waltham, USA). MgSO4 was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). CaCl2 and methanol were purchased from Aencore Chem. Pty. Ltd. (City of Whitehorse, Australia). Chloramphenicol was acquired from Bio Basic Inc. (Markham, Canada). Glucose was purchased from Showa Chemical Industry Co., Ltd. (Tokyo, Japan). Tris buffer was obtained from MDBio, Inc. (New Taipei City, Taiwan). [13C3]-pyruvate, [13C3]-lactate, [13C4]-succinate, [13C4]-fumarate, [13C4]-malate, [1,5,6-carboxyl-13C3]-citrate and 13C-formate were sourced from Cambridge Isotope Laboratories, Inc. (Tewksbury, USA). BCl3-methanol solution was purchased from Supelco (Merck Group, Bellefonte, USA). Sodium chloride was obtained from Panreac Química SLU (Barcelona, Spain). HCl was purchased from Biosynth Ltd. (Staad, Switzerland). Formic acid was acquired from J. T. Baker (Mallinckrodt Baker, Inc., Phillipsburg, USA). N, O-bis-[trimethylsilyl] trifluoroacetamide 1% trimethylchlorosilane was sourced from Pierce Biotech (Thermo Fisher Scientific Inc., Waltham, USA). Acetonitrile, n-hexane and acetone were purchased from ECHO Chemicals Co. Ltd. (Miaoli County, Taiwan). DB-225MS, DB-225 J&W GC, CP-Sil and HP-5MS columns were obtained from Agilent Technologies Ltd. (Santa Clara, USA).
8
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted using the Trizol Reagent (AG21102, Accurate Biology, China). To extract HCC tissues total RNA, 100 mg of tissue was weighed and finely minced, then 1 mL of Trizol Reagent (AG21102, Accurate Biology) was added. The mixture was grounded using a Tissue Homogenizer (LUKYM24, Lu Ka, China), followed by centrifugation at 1500 rpm for 15 min. The supernatant was carefully collected. For cell sample, cells were collected and washed twice with PBS. Each 1 × 106 cells were treated with 1 mL Trizol Reagent (AG21102, Accurate Biology). Then 0.2 mL of chloroform (C2432, Sigma-Aldrich, USA) was added to supernatant, and the mixture was vigorously shaken for 15 s. After 3 min incubation, the samples were centrifuged at 12,000 g for 15 min at 4 °C. The aqueous phase was carefully transferred to a new tube, and was precipitated by adding 0.5 mL of isopropanol. After 10 min incubation, the samples were centrifuged at 12,000 g for 10 min at 4 °C. The RNA pellet was washed with 75% ethanol (E809056, Macklin, USA), centrifuged again, and air-dried for 5–10 min. The RNA pellet was then dissolved in RNase-free water for further analysis. RNA samples went through qRT-PCR using Evo M-MLV RT kit (AG11711, Accurate Biology). cDNA samples for quantitative Real-Time PCR were prepared using a mix of SYBR Green Pro Taq HS qPCR kit (AG11701, Accurate Biology) and the primers listed in Table S3.
9
DNA and RNA Isolation from Biological Samples
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For sample preparation, the specimens were washed twice in phosphate-buffered saline (PBS) before DNA or RNA isolation. DNA was extracted from tissues or cultured cells by proteinase K digestion in combination with the DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer’s protocol. RNA was isolated using Trizol method. Each sample was resuspended in 500 μL Trizol (Thermo Fisher Scientific), briefly vortexed, and 100 μL chloroform (Sigma-Aldrich) was added. Phase separation was achieved by centrifuging the sample at 12,000 × g for 15 min at 4°C, then 200 μL of the aqueous phase was carefully transferred to a new tube. The RNA precipitation was carried out with 200 μL isopropanol (Sigma-Aldrich) and the RNA pellet was washed with 1 mL of 70% ice-cold ethanol.
10
Electrospinning of TIMP-1 Enriched Scaffolds
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The solutions were prepared 1 to 3 days before electrospinning. For each scaffold, a polyethylene glycol (PEG) (35 kDa, Sigma-Aldrich, Steinheim, Germany #81310) solution was prepared by adding 1.5 g of PEG and 3.5 g of chloroform (Sigma-Aldrich, Germany, #132950). The DP solution was produced by adding 0.6 g of DP powder, 3.52 g chloroform, and 0.88 g of 1,1,1,3,3,3-Hexafluoro-2-propanol (HFP, Sigma-Aldrich, Germany, #105228) into a glass with screw cap. For the incorporation of recombinant human TIMP-1 (PeproTech, Boston, MA, USA, #100-11-100UG), a total amount of 8 µg TIMP-1 dissolved in 400 µL phosphate-buffered solution (PBS, BioConcept, Allschwil, Switzerland, #3-05F39-I) containing rabbit serum albumin (RSA) (10 µg/mL TIMP-1 and 0.25% RSA in PBS) was added dropwise to the DP solution while stirring for 5 min at 500 rpm. The solution was shortly mixed on the vortex and further emulsified in an ultrasonic bath for 15 min. The emulsion was filled in a 5 mL glass syringe (Huberlab, Aesch, Switzerland, # 3.7102.33) and used immediately for electrospinning. As this protocol for water-in-oil emulsion with TIMP-1 was the same as used previously for PDGF-BB [24 (link)] and IGF-1 [15 (link)] incorporation, a homogenous distribution of water droplets containing TIMP-1 within the DP fibers can be assumed.
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