Female CD-1 mice (30 ± 3 g) were used for pilot IC experiments. IC was induced via i.p. injection of LPS (20 mg/kg from Escherichia coli (serotype 026:B6, Sigma-Aldrich, Oakville, ON, Canada) dissolved in 50 μL normal saline (Hospira, Montreal, Canada)). LPS was administered 15 min following induction of anesthesia, and the treatment compounds or vehicle (50 μL) administered i.p. 30 min following LPS administration. The four experimental groups for this model were as follows: (1) healthy control animals (CON; 50 μL normal saline i.p., treated with normal saline i.p., n = 9), (2) untreated IC animals (LPS; 50 μL LPS i.p., treated with normal saline i.p., n = 9), (3) IC treated with HU308 (LPS + HU308; 50 μL LPS i.p., treated with 5 mg/kg HU308 (Tocris Bioscience, Ellisville, MO, USA) dissolved in 30% DMSO, n = 4), (4) IC treated with BCP (LPS + BCP; 50 μL of LPS i.p., treated with 100 mg/kg i.p. BCP (Sigma-Aldrich, Oakville, ON, Canada) in normal saline; n = 7). Intravital microscopy (IVM) data was collected two hours following LPS administration for all animals used in this model.
Hu308
Manufactured by Bio-Techne
Sourced in United Kingdom, United States, Canada
HU308 is a lab equipment product from Bio-Techne. It is a high-performance device designed for laboratory applications. The core function of HU308 is to provide precise and reliable measurements for various research and analytical tasks.
Automatically generated - may contain errors
Lab products found in correlation
Jwh133, by Bio-Techne (3 mentions)
Sr141716a, by Bio-Techne (2 mentions)
Sr144528, by Cayman Chemical (2 mentions)
Sr144528, by Bio-Techne (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Am630, by Bio-Techne (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Escherichia coli, by Merck Group (1 mentions)
Normal saline, by Pfizer (1 mentions)
Normal saline, by Bio-Techne (1 mentions)
Normal saline, by Merck Group (1 mentions)
Dextran sulfate sodium (dss), by Merck Group (1 mentions)
Adenosine deaminase, by Merck Group (1 mentions)
192igg saporin, by Merck Group (1 mentions)
Guanosine 5 diphosphate, by Merck Group (1 mentions)
Cp55940, by Merck Group (1 mentions)
14c microscales, by American Radiolabeled Chemicals (1 mentions)
Biomax mr β radiation sensitivefilms, by Kodak (1 mentions)
Guanosine 5 o 3 thiotriphosphate gtpγs, by Merck Group (1 mentions)
18 protocols using hu308
1
Evaluating LPS-Induced Inflammation in Mice
2
Intravital Imaging of Lung Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipopolysaccharide (LPS) from Pseudomonas aeruginosa was purchased from Sigma-Aldrich (Oakville, ON, Canada) and dissolved in sterile saline (0.9% NaCl) to a concentration of 10 mg/mL prior to storage at −20 °C. HU-308 was purchased from Tocris Bioscience (Toronto, ON, Canada) and dissolved in anhydrous ethanol at 20 mg/mL prior to preparation in 1:1:18 solution of HU-308: Kolliphor EL: sterile saline and stored at 4 °C. Dexamethasone (Sandoz; Basel, Switzerland) was dissolved in sterile saline to a concentration of 33.3 µg/mL and stored at room temperature. Albumin-fluorescein isothiocyanate (FITC-albumin; λEX = 450–490 nm; Sigma-Aldrich) was dissolved in sterile saline to 50 mg/mL. Rhodamine-6G (λEX = 515–560 nm; Sigma-Aldrich) was dissolved in sterile saline to 0.5 mg/mL. Sodium pentobarbital (240 mg/mL; BimedaMTC Animal Health Inc.; Cambridge, ON, Canada) was diluted with sterile saline and used to provide anesthesia or euthanasia, where applicable. Isoflurane USP (99.9%; Fresenius Kabi; Bad Homburg, Germany) was used to provide inhalant anesthesia for lung intravital experiments only.
3
Isolation and Stimulation of Peritoneal Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice peritoneal macrophages were collected as described previously [15 (link)–17 (link)]. In brief, CB2R KO mice and age- and sex-matched WT controls were injected 10% thioglycollate (i.p.) for 3 days. Resident peritoneal macrophages were collected from the peritoneal cavity by flushing with 5 mL of ice-cold Hanks’ balanced salt solution containing 10 U/mL heparin. Macrophages were plated at a density of 5×104/mL in Dulbecco’s modified essential medium supplemented with 10% fetal calf serum, and were left to adhere for 4 hours in a humidified atmosphere at 37°C with 5% CO2. Then cells were washed twice, and the remaining cells were primed with 10 ng/ml lipopolysaccharides (LPS, Sigma, Louis, MO, USA) for 1 h, and then were stimulated with 3% DSS (mol. wt. 36,000 to 50,000 kDa, MP Biomedicals LLC, Santa Ana, CA, USA) in the presence or absence of 10 μM HU 308 (TOCRIS Bioscience, Bristol, BS, UK) for 24 h [18 (link),19 (link)]. In another set of experiments, peritoneal macrophages from WT and CB2R KO mice were stimulated with/without LPS/DSS for 24 h.
4
Cannabinoid Receptor Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
192IgG-saporin was acquired from Millipore
(Temecula, CA, USA). [35S]GTPγS (1250 Ci/mmol) and
[3H]CP55,940 (131.8 Ci/mmol) were acquired from PerkinElmer
(Boston MA, USA). The [14C]-microscales, used as standards
in the autoradiographic experiments, were acquired from American Radiolabeled
Chemicals (St. Louis, MO, USA). Kodak Biomax MR β-radiation-sensitive
films, bovine serum albumin (BSA),dl -dithiothreitol, adenosine
deaminase, guanosine 5′-diphosphate, guanosine 5′-O-3-thiotriphosphate
(GTPγS), ketamine and xylazine, as well as CP55,940 were acquired
from Sigma-Aldrich (St. Louis, MO, USA). SR141716A and HU308 were
acquired from Tocris Bioscience (Bristol, UK) and SR144528 from Cayman-Chemicals
(MI, USA). All the compounds used were of the highest commercially
available quality.
(Temecula, CA, USA). [35S]GTPγS (1250 Ci/mmol) and
[3H]CP55,940 (131.8 Ci/mmol) were acquired from PerkinElmer
(Boston MA, USA). The [14C]-microscales, used as standards
in the autoradiographic experiments, were acquired from American Radiolabeled
Chemicals (St. Louis, MO, USA). Kodak Biomax MR β-radiation-sensitive
films, bovine serum albumin (BSA),
deaminase, guanosine 5′-diphosphate, guanosine 5′-O-3-thiotriphosphate
(GTPγS), ketamine and xylazine, as well as CP55,940 were acquired
from Sigma-Aldrich (St. Louis, MO, USA). SR141716A and HU308 were
acquired from Tocris Bioscience (Bristol, UK) and SR144528 from Cayman-Chemicals
(MI, USA). All the compounds used were of the highest commercially
available quality.
5
Ligand Binding and Cell Signaling Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
G418 was purchased from KSE Scientific (Durham, NC). Puromycin, DMEM, penicillin/streptomycin, gentamicin, DPBS, Hank’s Buffer, HEPES, EDTA, Tris base, sucrose, MgCl2, Millipore filter plates and punch kits, EcoLite scintillation cocktail, and Poly-d -lysine coated 96-well plates were purchased from Fisher Scientific (Waltham, MA). Ambisome and FBS were purchased from Atlanta Biologicals (Flowery Branch, GA). Ro 20-1724, acetonitrile, DMSO, lipopolysaccharide (LPS), polyethyleneamine, and fatty acid-free BSA were purchased from Sigma Aldrich (St. Louis, MO). Antibodies against murine CD16/32 (ab33550) and CD206 (ab64693) were purchased from Abcam (Cambridge, UK). High bind plates (L15XB-3), anti-rat (R32AA-5), and anti-rabbit (R32AB-1) SULFO-TAG antibodies were purchased from Meso-Scale Discovery (Gaithersburg, MD). Cellular dyes phalloidin and Hoechst were purchased from Cellomics (Pittsburgh, PA), and ACTOne Membrane Potential Dye was purchased from Codex BioSolutions (Gaithersburg, MD). Forskolin, CP 55,940, HU 308, JWH 133, and SR 144528 were purchased from Tocris (Bristol, UK). Tritiated CP 55,940 was purchased from PerkinElmer (Waltham, MA). The 96-well filter plates and punch tips were purchased from Millipore (Billerica, MA).
6
Diabetic Cardiomyopathy Mice Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Diabetic cardiomyopathy mice model was created by intraperitoneal injection of streptozotocin (STZ) (dissolved in 0.1 mol/L citrate buffer, pH 4.5) at the dose of 50 mg/kg body weight per day for five consecutive days as previously described (Zhang et al., 2017 (link); Xiao et al., 2018 (link)). Citrate buffer without STZ loading was injected to mice in an equal volume as control. One week after the final STZ injection, fasting blood glucose was consecutively detected twice and the mean value was calculated. Only those with mean value of fasting blood glucose more than 16.6 mmol/L were considered as diabetic mice. For certain groups, mice were treated with HU308 (3 mg/kg body weight, TOCRIS Bioscience, Bristol, United Kingdom), a specific agonist for CB2 (Hanus et al., 1999 (link)), and/or bafilomycin A1 (0.3 mg/kg body weight, Selleckchem, Houston, TX, United States) every day from 12 to 14 weeks after the final injection of STZ. Normal saline in same volume was injected as vehicle.
7
CB2 Receptor Agonists and AKT Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
JWH-133 and HU-308 were purchased from Tocris Cookson (Bristol, UK), and the CB2 antagonist SR 144528 was from Cayman Chemical (Ann Arbor, MI). The drugs were dissolved in dimethylsulfoxide (DMSO). Antibody anti-phospho-AKT (Ser473), anti-total-AKT and anti-phospho-GSK3β (Ser9) were obtained from Cell Signaling Technology (St Louis, MO, USA). We purchased antibodies anti-SNAIL, anti-β-Actin, and AKT Inhibitor (AKTi-1/2) from Abcam (Cambridge, UK). Anti-E-cadherin antibody is from Becton Dickinson (San Jose, CA, USA) and anti-β-catenin antibody is from Dako (Glostrup, Denmark).
8
CB2 Receptor Agonist Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
9
Dose-dependent CB2 Receptor Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animals were randomly assigned to experimental groups after a 1-week habituation and 1-week handling periods. A dose-response for CB2R ligands (0.5 and 5 mg/kg) was performed considering previous pharmacological ranges [25 (link), 28 (link), 29 (link), 45 (link)]. The CB2R agonist HU308 (Tocris, Bristol, UK; abbreviated nomenclature: HU) or the inverse agonist/antagonist AM630 (Tocris, Bristol, UK; abbreviated nomenclature: AM) were administered for 2 weeks. Unpredictable chronic mild stress (uCMS) was induced as previously described [46 (link)–48 (link)]. Briefly, animals were subjected to an 8-week unpredictable chronic stress paradigm followed by a 2-week milder protocol period during which CB2R ligands were administered in combination with a PE protocol as previously described [49 (link)]. Four weeks before sacrifice, all animals received intraperitoneal (i.p.) injections of BrdU (50 mg/kg; Sigma Aldrich, MO, USA) for 5 consecutive days (twice per day) to evaluate the survival of newborn neurons. Further details are described in the supplementary information.
10
Synthesis and Preparation of Cannabinoid Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SR141716A was synthesized by IRG, University of Aberdeen (according to US Patent 5,462,960). (R)(-)-DOI hydrochloride (CAS 82864–02-6), DMSO, and Kolliphor® EL were from Sigma-Aldrich (Rehovot, Israel). Ethanol was from Merck, Germany. HU-308 was from Tocris, UK. E-BCP was from Kanata Enterprises, India (99%). Δ9-THC (98%) was kindly provided by Prof. Mechoulam (The Hebrew University, Israel). DOI (1 mg/kg) was dissolved in saline. HU-308 (0.2 mg/kg, 1 mg/kg, 5 mg/kg), E-BCP (1 mg/kg, 5 mg/kg, 10 mg/kg), and Δ9-THC (5 mg/kg) were dissolved in vehicle made of 0.6:1:1.84 DMSO: Kolliphor® EL:saline. SR141716A (5 mg/kg, 10 mg/kg, 20 mg/kg) was dissolved in vehicles 0.6:1:1.84 ethanol: Kolliphor® EL: saline or DMSO: Kolliphor® EL:saline, as indicated in legends. The drugs were freshly prepared, aliquoted, and stored at − 20 ℃ for up to 3 months. Each aliquot was discarded after one use. Drugs were injected intraperitoneally (i.p.). All injections were made in a volume of 10 µl/g.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!