Platinum-cured silicone tubes (Cole Parmer) with inner diameters of 250–1000 μm were used as a mold for the core. Precursor solution composed of 80% wt vol−1 PEGDA (700 Da; Sigma Aldrich), 5% wt vol−1 2-hydroxy-2-methyl-propiophenone (Sigma Aldrich) in distilled water was injected in the tube through a syringe adapted with a syringe filter with 0.45 μm pore. The PEG hydrogel was formed by photocrosslinking the solution with exposure to UV (365 nm, 5 mW cm−2; Spectroline) for 5 min. The tube with the crosslinked core was immersed in dichloromethane for 30 min, and then the core was isolated from the swollen tube. The core was immersed in distilled water at least for 1 hour to remove unreacted chemicals. To form the clad layer, the core was immersed in alginate solution (2 % wt vol−1; Sigma Aldrich) and then in calcium chloride solution (100 mM; Sigma Aldrich). This procedure was repeated to form a multi-layer clad. Successful fabrication of the core-clad fiber was checked by phase-contrast microscopy (Olympus).
Calcium chloride solution
Manufactured by Merck Group
Sourced in United States
Calcium chloride solution is a clear, colorless liquid chemical compound consisting of calcium and chloride ions in an aqueous solution. It is a common laboratory reagent used for various analytical and experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
Alginate solution, by Merck Group (1 mentions)
2 hydroxy 2 methylpropiophenone, by Merck Group (1 mentions)
Pegda, by Merck Group (1 mentions)
Phase contrast microscopy, by Olympus (1 mentions)
Vitamin d3, by Merck Group (1 mentions)
Mgso4 7h2o, by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Kh2po4, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
D luciferin, by Duchefa Biochemie (1 mentions)
Bovine thrombin, by Merck Group (1 mentions)
Sodium alginate, by Merck Group (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
100 m cell strainer, by Corning (1 mentions)
Tris edta, by Merck Group (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Sodium alginate solution, by Merck Group (1 mentions)
7 protocols using calcium chloride solution
1
Fabrication of Hydrogel Optical Fibers
2
Calcium, Vitamin D3 Supplementation in Rats
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Provision of calcium and vitamin D3 was conducted for 4 weeks (Fig 1A ). Rats were administered ad libitum with 15 mM calcium chloride solution (catalog no. 21114, Sigma Chemical Co., St. Louis, MO, USA.) and 12 mM monosaccharides (i.e., glucose; catalog no. 1009378, Ajax Finechem Pty Limited, New South Wales, Australia and galactose; catalog no. 0802339, Asia Pacific Specialty Chemicals Ltd., New South Wales, Australia) and 46.5 mM sodium chloride (catalog no. S5068-1-1000, Quality Reagent Chemical (QrëcTM, New Zealand) according to the formula of Suntornsaratoon et al. (2015) [35 (link)]. Distilled water (solvent) or calcium chloride solution was added in drinking bottles for daily treatment.
Vitamin D3 (catalog no. C9756, Sigma Chemical) was dissolved in sterile soybean oil (Thanakorn Vegetable Oil Products Co., Ltd., Thailand) and given orally at 400 IU/kg body weight of rats (soybean oil volume of 0.3 mL), as previously reported by Vieth (2004) and Detregiachi et al. (2016) [36 (link),37 (link)].
Vitamin D3 (catalog no. C9756, Sigma Chemical) was dissolved in sterile soybean oil (Thanakorn Vegetable Oil Products Co., Ltd., Thailand) and given orally at 400 IU/kg body weight of rats (soybean oil volume of 0.3 mL), as previously reported by Vieth (2004) and Detregiachi et al. (2016) [36 (link),37 (link)].
3
Mitochondrial ATP Dynamics Quantification
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Twelve hours after transfection with Mitochondrial-targeted luciferase enzyme (mtLUC), cells were re-plated into a 96-multiwell White/Clear Bottom Plate, TC Surface (Thermo Fisher) (10x103 cells/well) for the luminescence measurements by a PerkinElmer EnVision plate reader equipped with two injector units. For the ATP measurements, cells were washed twice with a modified Krebs Ringer Buffer (KRB: 135mM NaCl (Sigma-Aldrich), 5mM KCl (Sigma-Aldrich), 0.4mM KH2PO4 (Sigma-Aldrich), 1mM MgSO4(7 H2O) (Sigma-Aldrich), 1mM MgCl2 (Sigma-Aldrich), 20mM HEPES (Sigma-Aldrich), pH 7.4), glucose (0.1% D-(+)-Glucose (Sigma-Aldrich))/Ca2+ (1mM Calcium chloride solution (Sigma-Aldrich) and placed in 50 μL of KRB + glucose/Ca2+. After recording the basal background signal for 5 s (s), a 20 μM (final concentration) D-luciferin (Duchefa Biochemie) solution in KRB + glucose/Ca2+ was added and, since luciferin is cell-permeable, the luminescence was detected immediately. After 100 s, when the signal reached a plateau, a 100 μM (final concentration) histamine solution in KRB + glucose/Ca2+ with 20 μM (final concentration) D-luciferin was added to boost ATP production. For each measurement, the luminescence signal (CPS, counts per second) after histamine was normalized on the mean CPS at the plateau reached after the first luciferin addition.
4
Autologous PRP Preparation Technique
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PRP was prepared using a technique described by Okuda et al. (23 (link)). Briefly, 60 mL of autologous blood withdrawn from each dog was initially centrifuged at 2400 rpm for 10 minutes to separate PRP and PPP portions from the red blood cell fraction. The PRP and PPP portions were again centrifuged at 3600 rpm for 15 minutes to separate PRP from PPP. The resulting pellet of platelets was resuspended in 3.0 ml of residual plasma. The PRP was activated at the time of application with a 10% calcium chloride solution (Sigma, USA) and 5000 U of bovine thrombin (Sigma, USA).
5
Functional Food Capsule Development
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For the capsule development reagents, sodium alginate, DPPH, and calcium chloride solution were purchased from Sigma Aldrich Company, Romanian branch. Natural juice from organically certified aronia was provided by SC Andy Star SRL. According to the producer, 100 mL of chokeberry juice contains 0.11 g protein, 15.75 g carbohydrates (from which 6.49 g are natural sugars), 0.5 g fibers, and 0.0016 g salt. The energy value of the product is 64.88 kcal/100 mL juice.
Juice from organic sea buckthorn was provided by SC NATUR LOGISTICSSRL; 100 mL of juice contains 1 g fats (from which 0.2 g saturated fatty acids), 4 g carbohydrates (from which 4 g simple sugars), less than 0.8 g proteins and less than 0.01 g salt. The total energy value is 39 kcal/100 mL juice.
Blueberry juice was obtained in our laboratory by fresh fruit cold-pressing and its nutritional values are less than 0.5 g fat, 10 g carbohydrates, 9.2 g sugars, less than 0.5 g fibers, less than 0.5 g proteins, and less than 0.01 g salt. The energetic value of 100 mL of juice was 41 kcal.
For yogurt preparation we used fresh raw cow milk with 3.5% fat, 3% protein and 4.5% carbohydrates content, from our local producers. Lactic bacteria such as Lactobacillus bulgaricus and Streptococcus thermophilus were provided from SC Enzyme & Derivates SA Romania.
Juice from organic sea buckthorn was provided by SC NATUR LOGISTICSSRL; 100 mL of juice contains 1 g fats (from which 0.2 g saturated fatty acids), 4 g carbohydrates (from which 4 g simple sugars), less than 0.8 g proteins and less than 0.01 g salt. The total energy value is 39 kcal/100 mL juice.
Blueberry juice was obtained in our laboratory by fresh fruit cold-pressing and its nutritional values are less than 0.5 g fat, 10 g carbohydrates, 9.2 g sugars, less than 0.5 g fibers, less than 0.5 g proteins, and less than 0.01 g salt. The energetic value of 100 mL of juice was 41 kcal.
For yogurt preparation we used fresh raw cow milk with 3.5% fat, 3% protein and 4.5% carbohydrates content, from our local producers. Lactic bacteria such as Lactobacillus bulgaricus and Streptococcus thermophilus were provided from SC Enzyme & Derivates SA Romania.
6
Isolation of Primary Human Hepatocytes
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Human liver tissue fragments were obtained and isolated from 3 hepatectomy patients who underwent surgical treatment for various reasons at Hanyang University Medical Center in Seoul, Korea (Supplementary Table 1 ). The isolation of mouse primary hepatocytes or hPHs followed a previously published method [2 (link)17 (link)23 (link)]. For mouse samples, the livers were perfused in vivo through a portal vein. For human samples, hPHs were isolated with a perfusion pump (BT100-1F, Dongbang Hitech) using 2-step perfusion as follows; first, perfuse the sample with warmed liver perfusion solution supplemented with tris-EDTA (Sigma-Aldrich); second, continue with enzymatic digestion by perfusion of collagenase/elastase mixture (Worthington Biochemical) and calcium chloride solution (Sigma-Aldrich). Next, the digested tissue was minced with a surgical blade and filtered with a 100-µm cell strainer (Corning) to isolate the single-cell hPH suspension. Lastly, viable and live hPHs were sorted by 25% Percoll (GE Healthcare) isodensity centrifugation and seeded on a 10-cm2 dish collagen-coated plate (Advanced BioMatrix) in William’s E Media Gibco with Primary Hepatocyte Maintenance Supplement (Gibco) supplementation.
7
3D Printed Dual-Drug Scaffolds
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To fabricate the dual-drug-based scaffolds, a drug-loaded scaffold was first prepared by 3D printing, as reported previously [12 (link)]. Briefly, PCL (MW: 45,000; Sigma-Aldrich, St Louis, MO, USA) and cefazolin (CFZ) (Chong Kun Dang pharmaceutical Corp., Seoul, Korea) were mixed at concentrations of 25 mg/mL and 50 mg/mL, respectively, and then ejected using a 3D bioprinting system (Geo Technology, Incheon, Korea) to a 200 μm nozzle (inner diameter) supplied with 800 kPa pressure at a constant speed of 100 mm/min. The resultant cefazolin-loaded scaffolds were cut into 5 mm disks using a punch and immersed in another 12-well plate containing 4% sodium alginate solution (Sigma-Aldrich, St Louis, MO, USA) blended with 9 wt% rifampicin (RFP) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and 100 mM calcium chloride solution (Sigma-Aldrich, St Louis, MO, USA) was added for 30 s at room temperature to encapsulate alginate. The fabrication process of the dual-drug-based scaffold is described in Figure 1 A.
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