Cannabidiol (2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol) was obtained from Tocris Bioscience (Bio-Techne, Abingdon, United Kingdom). AM281 (CB1 receptor antagonist; 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and AM630 (CB2 receptor antagonist; 6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone) were obtained from Cayman Chemicals (Ann Arbor, MI). SB-366791 (N-(3-methoxyphenyl)-4-chlorocinnamide), rhodamine 6G, cremophor, dimethyl sulphoxide (DMSO), urethane, and MIA were obtained from Sigma-Aldrich (St. Louis, MO). Solutions of CBD, AM281, AM630, and SB-366791 were prepared in vehicle (1:1:18; DMSO:cremophor:saline) on the day of use. rhodamine 6G (0.05%) and MIA were dissolved in saline. Physiological buffer (135 mM NaCl, 20 mM NaHCO3, 5 mM KCl, 1 mM MgSO4*7H2O, pH = 7.4) was prepared in the laboratory.
Cannabidiol
Manufactured by Bio-Techne
Sourced in United Kingdom
Cannabidiol is a pure compound extracted from the hemp plant. It is a colorless crystalline solid that is highly soluble in organic solvents. Cannabidiol is commonly used as a reference standard in analytical testing and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulphoxide, by Merck Group (1 mentions)
Cremophor, by Merck Group (1 mentions)
Rhodamine 6g, by Merck Group (1 mentions)
Sb366791, by Merck Group (1 mentions)
Am281, by Cayman Chemical (1 mentions)
Am630, by Cayman Chemical (1 mentions)
Tranilast, by Merck Group (1 mentions)
δ9 tetrahydrocannabinol thc, by Merck Group (1 mentions)
Tissue culture reagents, by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Rpmi medium, by Lonza (1 mentions)
Anandamide, by Cayman Chemical (1 mentions)
Hu210, by Bio-Techne (1 mentions)
Hu 211, by Cayman Chemical (1 mentions)
Mitoxantrone, by Merck Group (1 mentions)
14 protocols using cannabidiol
1
CBD Effects on Neuronal Activity
2
CBD, THC, and Tranilast Protocol
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Cannabidiol (CBD) was purchased from Tocris Cookson (Bristol, UK). Tissue culture reagents were purchased from Gibco-BRL (San Giuliano Milanese, MI, Italy) and Sigma (St. Louis, MO, USA). Δ9-tetrahydrocannabinol (THC) and tranilast (TNL) were purchased from Sigma. The authorization number for the scientific use of THC was n. SP/94, of 07.07.2021.
3
CBD and Doxorubicin Combination Treatment
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Cannabidiol was obtained from Tocris Bioscience (Bristol, UK). CBD was initially dissolved in DMSO (stock concentration of 75 mM) with subsequent dilutions in serum-free tissue culture medium for in vitro experiments. Doxorubicin hydrochloride was purchased from Abcam (Cambridge, UK) and the stock dilution in DMSO to 25 mM was diluted in serum-free medium. The following antibodies were purchased from Cell Signaling Technology [New England Biolabs (NEB), Hertfordshire, UK]: PathScan® Multiplex Western Cocktail I (#5301), anti-Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (#5536), β-Actin (8H10D10) Mouse mAb (#3700), anti-Rabbit IgG, HRP-linked Antibody (#7074) and anti-Mouse IgG, HRP-linked Antibody (#7076). Concanavalin A was from Sigma–Aldrich, UK (#C2272) with stock solutions stored at 5 mg/mL in dH2O.
4
Isolation and Treatment of Glial Cells
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Primary microglia and astrocytes were isolated and cultured from C57BL/6J and CX3CR1GFP/wt [51 (link),52 (link)] mice as described before [53 (link),54 (link)]. After 10–14 days microglial cells were isolated from astrocytic monolayer and used for further experiments. A375 [55 (link)] (gifted by Simon Jasinski-Bergner, University Halle-Wittenberg, Halle (Saale), Germany), BV2 microglia [56 (link)] (obtained from Ullrich, University of Zürich, Zürich, Switzerland) and primary glial cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). LN229 [57 (link)] were cultured in RPMI medium (Lonza, Basel, Switzerland) with 10% FBS and 1% penicillin/streptomycin. After two days, the medium was collected from confluent astrocytes or BV2 microglia, filtered (Sarstedt, Nümbrecht, Germany), and applied on the tumor cells in a 1:1 ratio with the respective culture medium. This medium was added 3 h before starting the imaging for both single cell and collective migration experiments.
For cannabinoid treatment cannabidiol (5 µM, Tocris Bioscience, Bristol, UK) [39 (link),58 (link),59 (link)], tetrahydrocannabinol (5 µM, Tocris) [42 (link)] or a combination of both was applied 3 h before the start of the experiments. THC and CBD were both dissolved in DMSO.
For cannabinoid treatment cannabidiol (5 µM, Tocris Bioscience, Bristol, UK) [39 (link),58 (link),59 (link)], tetrahydrocannabinol (5 µM, Tocris) [42 (link)] or a combination of both was applied 3 h before the start of the experiments. THC and CBD were both dissolved in DMSO.
5
Cannabidiol Intracerebral Microinjection Protocol
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Cannabidiol (Tocris, UK) was dissolved in a mixture of dimethylsulfoxide (DMSO; 10%) and phosphate buffer solution (PBS, 90%) (Shirazi-zand, Ahmad-Molaei, Motamedi, & Naderi, 2013 (link)). Microinjection of Cannabidiol into right lateral ventricle was in a volume of 2 μL. ICV injection of Cannabidiol at doses of 50, 100, and 200 ng/rat (Shirazi-zand et al., 2013 (link)) was given by inserting a 30-gauge injection cannula 1 mm beyond the tip of the guide cannula to the site of injection. The injection cannula was connected with a polyethylene tube to a Hamilton syringe. Injection (1μL) was done over 60s. As, Cannabidiol was injected in the treatment groups, the vehicle group received DMSO 10% through ICV injection. Analysis between control and vehicle groups was done because there are evidences about possible neuroprotective role of vehicle (dimethylsulfoxide) (Di Giorgio et al., 2008 (link)).
6
Cannabinoid Receptor Ligand Procurement
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Cannabidiol (2-((1S,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-2-enyl)-5-pentylbenzene-1,3-diol), and HU 210 ((6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,0a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b.d]pyran-9-methanol) were obtained from Tocris Cookson (Bristol, UK). Anandamide (N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide), and HU 211 (3-(1,1-dimethylheptyl)-6aS,7,10,10aS- tetrahydro-1-hydroxy-6,6-dimethyl-6H- dibenzo[b,d]pyran-9-methanol; dexanabinol) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). DMSO (dimethyl sulfoxide) were obtained from Sigma-Aldrich Sweden AB (Stockholm, Sweden). Alcohol prep pads (70% isopropyl alcohol) and saline were from Apoteket AB (Umeå, Sweden).
7
Preparation of Cytotoxic Drug Solutions
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Cisplatin (CDDP—Sigma-Aldrich) was dissolved in phosphate buffered saline (PBS) with Ca2+ and Mg2+. Mitoxantrone (MTX—Sigma-Aldrich) was dissolved in DMSO as stock solutions. The cannabidiol (CBD—Tocris, Bristol, UK) was dissolved in ethanol as stock solutions in concentration of 5 mg/mL. The drugs were dissolved to the respective concentrations with culture medium before use.
8
Cannabidiol Oocyte Treatment Protocol
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First, 10 mM cannabidiol (CBD; Tocris, Bristol, UK) stocks were prepared and were stored at −20 °C. Directly before the CBD treatment for oocytes started, stocks were diluted with oocyte Ringer (ORi) to result in concentrations of 20 μM and 40 μM.
9
Synthesis and Use of Cannabinoids
10
Neuroprotective Compound Screening in PCD Model
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Approaches 1 and 2 (Figure 1 B ): To evaluate the neuroprotective potential of compounds that modulate calcium‐sensitive mitochondrial‐associated signalling, we co‐applied the compounds with anti‐Yo/rCDR immediately at PCD onset (approach 1) or 4 days after PCD induction (approach 2); cultures were treated for 6 days. Compounds tested were AM281 [IC50: 12 nM (CB1R), 4.2 μM (CB2R); #1115, Tocris Bioscience, Bristol, UK], benzamil [IC50: approximately 100 nM (NCX); #3380; Tocris], butylated hydroxytoluene [BHT; IC50: 3.5 μM (ROS); #W218405; Sigma], cannabidiol [CBD; IC50: 3.35 μM (CB1R), 27.5 μM (CB2R); #1570; Tocris], CGP37157 [IC50: 400 nM (NCX); #1114; Tocris], cyclosporin‐A [CsA; IC50: 5 nM (MPTP); #1101; Tocris] and KB‐R7943 [IC50: 700 nM (NCXrev), 5.5 μM (MCU); #1244; Tocris]. As this PCD model system is based on an interface hydrophilic PTFE membrane‐diffusion approach where the cOTSC is not submerged, the system behaves like an in vivo brain. Therefore, several test dosages were evaluated starting with 2.5‐fold of the IC50 value to determine effective, nontoxic doses if there was no literature guided initial dose selection available for this kind of culture system. Depending on the hydrophilic or hydrophobic nature of the used compounds, the effective dosage was determined between 2.5 to 200 times of the IC50 value.
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