After baseline testing (behavior) or at the start of the experiment, animals were anesthetized with isoflurane (5% induction; 1%-2% maintenance). A Hamilton syringe attached to calibrated polyethylene tubing and a 30-gauge needle were used to administer agents subcutaneously to the midplantar hind paw. Vehicle (1% DMSO) or a PI3K isoform-specific antagonist and 100 μL, 2% λ-carrageenan (Wako Pure Chemical Industries, Ltd) contained in the same tubing were injected 2 minutes apart. Class 1 PI3K antagonists used were: compound 15-e (mW 313.4; Enzo Life Sciences, Farmington, NY), alpha; TGX 221, (mW 364.4; Cayman Chemical Company, Ann Arbor, MI), beta; Cal-101 (mW 415.42; Selleck Chemicals, Houston, TX), delta; and AS252424 (mW 305.28; Cayman Chemical Company), gamma. Aliquots of antagonists in DMSO were defrosted and diluted just before use. Vehicle and all antagonist solutions were adjusted to pH of 7.2 to 7.4. Despite different reported IC50s, starting or standard doses of each antagonist were 5 nm administered in 50 μL of vehicle. Doses used were based on recent work.13 (link),35 (link)
λ carrageenan
Manufactured by Fujifilm
Sourced in Japan
λ-carrageenan is a polysaccharide extracted from red seaweed. It is a common ingredient used in various laboratory applications as a thickening, gelling, and stabilizing agent.
Automatically generated - may contain errors
Lab products found in correlation
Cal 101, by Selleck Chemicals (1 mentions)
Compound15e, by Enzo Life Sciences (1 mentions)
As 252424, by Cayman Chemical (1 mentions)
Hyaluronic acid, by Fujifilm (1 mentions)
Heparin, by Nacalai Tesque (1 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
κ carrageenan, by Fujifilm (1 mentions)
4 protocols using λ carrageenan
1
PI3K Isoform-Specific Antagonist Effects
2
AGE-induced Modifications of Bovine Serum Albumin
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Bovine serum albumin (BSA; FUJIFILM Wako, Osaka, Japan) was incubated under sterile conditions with D-glyceraldehyde (AGE-2) (Sigma–Aldrich, St. Louis, MO, USA) or glycolaldehyde dimer (AGE-3) (Sigma–Aldrich) in 0.2-M phosphate buffer (pH 7.4) at 37°C for 7 days. As a control, BSA was incubated under the same conditions without additional compounds. After the incubation, AGE–BSA and BSA were dialysed for 2 days at 4°C. The endotoxin concentration of AGEs at 100 µg/mL was measured by SRL (Okayama, Japan) as 1.2 pg/mL. Fucoidan derived from Fucus vesiculosus (Sigma–Aldrich), dextran 500,000 (FUJIFILM Wako), low-molecular-weight (LMW) dextran sulphate (average MW, 5000; FUJIFILM Wako), high-molecular-weight (HMW) dextran sulphate (average MW, 500,000; FUJIFILM Wako), chondroitin sulphate C sodium salt (FUJIFILM Wako), heparin (Nacalai Tesque, Kyoto, Japan), hyaluronic acid (FUJIFILM Wako) and neocarrahexaose-24,41,3,5-tetra-O-sulphate (Dextra Laboratories, Reading, UK) were dissolved in ultrapure water. λ-carrageenan (FUJIFILM Wako) was dissolved in ultrapure water and incubated at 60°C for 30 min. All reagents were prepared under sterile conditions.
3
Abdominal Aortic Adventitial Inflammation
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To produce the abdominal aortic adventitial inflammation, we embedded carrageenan (a polysaccharide) around the isolated abdominal aorta segment (2.5 cm between inferior mesenteric artery and lumbar artery) as shown in Figure 1 . Carrageenan is a potent chemoattractant for monocytes and can induce the focal accumulation of inflammatory macrophages (Wang et al., 2004 (link)). For this undertaking, rabbits were anesthetized by intramuscular injection of ketamine (25 mg/kg BW) + medetomidine hydrochloride (.5 mg/kg BW) and the abdominal aortas were exposed. A piece of sterilized gauze was pre-soaked in 10 mg/mL of λ-carrageenan (Wako Chemicals, Osaka, Japan) in .9% NaCl (w/v) at 40°C overnight, laid around the isolated abdominal aorta without any arterial branches, and gently covered with a sheet of polyethylene film to prevent inflammatory adhesion to the surrounding tissue. All rabbits were sacrificed at 24 weeks after surgery by intravenous injection of an overdose of sodium pentobarbital solution. The whole abdominal aortas were carefully collected for histological examination, immunostaining, and RNA and protein extraction (see below). This study was approved by the Animal Care Committee of the University of Yamanashi and Saga University and conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH.
4
Harvesting Bacteria from Kappaphycus alvarezii
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ISSN: 2456-1878 https://dx.doi.org/10.22161/ijeab.61.12 82
Algae K. alvarezii were collected from USA Marine Biological Institute, Kochi University, Japan and floated in Uranouchi Bay, Tosa, Kochi Prefecture, Japan for one week to collect the bacteria. While several commercial carrageenan was used in this study, κ-carrageenan and λcarrageenan purchased from Wako.
Algae K. alvarezii were collected from USA Marine Biological Institute, Kochi University, Japan and floated in Uranouchi Bay, Tosa, Kochi Prefecture, Japan for one week to collect the bacteria. While several commercial carrageenan was used in this study, κ-carrageenan and λcarrageenan purchased from Wako.
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