The nineteen test agonists, N6-modified adenosine analogues, were synthesized and initially characterized pharmacologically at the hA3AR overexpressed in CHO cells, as previously reported [15 (link)–17 (link)]. Reference agonists 2-Cl-IB-MECA (2-chloro-N6-(3-iodobenzyl)-5′-N-methylcar-boxamidoadenosine) and NECA (5′-N-ethylcarboxamidoadenosine) were purchased from Tocris Bioscience (Biotechne, Abingdon, UK). Poly-D-lysine hydrobromide and DOTAP Liposomal Transfection Reagent were purchased from Sigma-Aldrich (Steinheim, Germany). FuGene® HD and Nano-Glo Live Cell Reagent were provided by Promega (Madison, WI, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with GlutaMAX®, Hank’s Balanced Salt Solution (HBSS), Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), pertussis toxin (PTX), penicillin/streptomycin (10,000 IU/mL and 10,000 μg/mL) and amphotericin B (250 μg/mL) were bought from Thermo Fisher Scientific (Pittsburg, PA, USA). The anti-dNGFR (truncated nerve growth factor receptor) antibody was purchased from Chromaprobe (Maryland Heights, MO, USA). Human Embryonic Kidney (HEK) 293T cells (passage 20) were a kind gift of Prof. O. De Wever of the Laboratory of Experimental Cancer Research (Ghent University Hospital, Belgium). The cell lines CB1-NanoBiT®-βarr2 and CB1-NanoBiT®-miniGαi were described before [14 ,18 (link),19 (link)].
5 n ethylcarboxamidoadenosine neca
Manufactured by Bio-Techne
Sourced in United Kingdom
5′-N-ethylcarboxamidoadenosine (NECA) is a synthetic adenosine receptor agonist. It is used as a research tool to study the pharmacological effects of adenosine receptor activation.
Automatically generated - may contain errors
Lab products found in correlation
Fugene hd, by Promega (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Nano glo live cell reagent, by Promega (1 mentions)
Poly d lysine hydrobromide, by Merck Group (1 mentions)
Dotap liposomal transfection reagent, by Merck Group (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by GE Healthcare (1 mentions)
Pertussis toxin ptx, by Merck Group (1 mentions)
3h adenine, by PerkinElmer (1 mentions)
14c camp, by PerkinElmer (1 mentions)
Alphascreen surefire p erk assay kit, by PerkinElmer (1 mentions)
L glutamine, by Lonza (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cytiva (1 mentions)
Oregon green 488 bapta 1 am, by Thermo Fisher Scientific (1 mentions)
U46619, by Cayman Chemical (1 mentions)
Enhanced chemiluminescence ecl substrate, by Cytiva (1 mentions)
9 protocols using 5 n ethylcarboxamidoadenosine neca
1
Characterization of N6-modified Adenosine Agonists
2
Purine Receptor Signaling Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
G418, Lipofectamine and Optimem were obtained from Invitrogen (Paisley, UK). FCS was obtained from PAA Laboratories (Wokingham, UK), L-glutamine from Lonza (Basel, Switzerland) and Fugene HD from Promega (Southampton, UK). Pertussis toxin (PTx) was purchased from Merck Chemicals (Nottingham, UK). NECA [5-(N-ethylcarboxamido)adenosine] and HEMADO [2-(1-hexynyl)-N-methyladenosine] were obtained from Tocris Bioscience (Bristol, UK). The AlphaScreen SureFire p-ERK assay kit was obtained from PerkinElmer (Waltham, MA, USA). [3H]-adenine and [14C]-cAMP were from Perkin-Elmer (Buckinghamshire, UK). CA200645 was purchased from Cellaura Technologies Ltd (Nottingham, UK). All other chemicals and reagents were purchased from Sigma-Aldrich (Gillingham, UK).
3
Platelet activation signaling pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Horm collagen was obtained from Takeda (High Wycombe, UK). NECA (5'-Nethylcarboxamidoadenosine) was purchased from Tocris Bioscience (Bristol, UK). Cangrelor was purchased from Medicines Company. U46619 was obtained from Cayman Chemical.
Chronolume and ATP were from Chrono-log Corporation (Manchester, UK). Horseradish peroxidase (HRP)-conjugated secondary antibodies and Enhanced Chemiluminescence (ECL) substrate were obtained from Amersham Biosciences (GE Healthcare, Bucks, UK). Oregon green 488 BAPTA-1-AM was purchased from Invitrogen (Invitrogen, ThermoFisher Scientific, Paisley, UK). Other reagents were obtained from Sigma.
Antibodies: PLCγ2 p1217, PLCγ2 p759, Syk p525/526, Syk p323, Syk p352, LAT p132 and LAT p171 were from Cell Signalling Technology (Danvers, Massachusetts, United States).
LAT p200 mAb was obtained from Abcam (Cambridge, UK). Mouse anti-human antiphosphotyrosine (clone 4G10) mAb was from Millipore UK Ltd (Watford, UK). Alexa Fluor 488 phalloidin and Alexa Fluor 647 were purchased from Invitrogen (Invitrogen, ThermoFisher Scientific, Paisley, UK). 1G5-Fab recognises monomeric and dimeric GPVI was raised as described 23 .
Chronolume and ATP were from Chrono-log Corporation (Manchester, UK). Horseradish peroxidase (HRP)-conjugated secondary antibodies and Enhanced Chemiluminescence (ECL) substrate were obtained from Amersham Biosciences (GE Healthcare, Bucks, UK). Oregon green 488 BAPTA-1-AM was purchased from Invitrogen (Invitrogen, ThermoFisher Scientific, Paisley, UK). Other reagents were obtained from Sigma.
Antibodies: PLCγ2 p1217, PLCγ2 p759, Syk p525/526, Syk p323, Syk p352, LAT p132 and LAT p171 were from Cell Signalling Technology (Danvers, Massachusetts, United States).
LAT p200 mAb was obtained from Abcam (Cambridge, UK). Mouse anti-human antiphosphotyrosine (clone 4G10) mAb was from Millipore UK Ltd (Watford, UK). Alexa Fluor 488 phalloidin and Alexa Fluor 647 were purchased from Invitrogen (Invitrogen, ThermoFisher Scientific, Paisley, UK). 1G5-Fab recognises monomeric and dimeric GPVI was raised as described 23 .
4
Adenosine Receptor Ligands and Analogs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The adenosine A1 receptor agonist N6-cyclopentyladenosine (N6-CPA, purity > 98%) was obtained from Abcam (Shanghai, China) and dissolved in DMSO at 50 mM. The adenosine A1 receptor antagonist rolofylline (KW-3902, purity > 99%) was obtained from Tocris Bioscience (Bristol, United Kingdom) and dissolved in DMSO at 10 mM. The adenosine A2a receptor agonist CGS 21680 (purity > 98%) was obtained from Abcam and dissolved in DMSO at 10 mM. The adenosine A2a receptor antagonist SCH 58261 (purity > 99%) was obtained from Abcam and dissolved in DMSO at 10 mM. The adenosine A2b receptor agonist BAY 60-6583 (purity > 98%) was obtained from Tocris Bioscience and dissolved in DMSO at 20 mM. The adenosine A2b receptor antagonist PSB603 (purity > 98%) was obtained from Tocris Bioscience and dissolved in DMSO at 10 mM. The adenosine A3 receptor agonist piclidenoson (IB-MECA, purity > 97%) was obtained from Abcam and dissolved in DMSO at 10 mM. The adenosine A3 receptor antagonist MRE 3008F20 (purity > 98%) was obtained from Tocris Bioscience and dissolved in DMSO at 50 mM. The adenosine analog 5′-N-ethylcarboxamidoadenosine (NECA, purity > 99%) was obtained from Tocris Bioscience and dissolved in DMSO at 50 mM. All solutions were stored at –20°C and used within 1 year.
5
Cellular Model for TGF-β Signaling
6
Calcium Signaling Pathway Modulation in Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMEM:F12 was obtained from Corning, and fetal bovine serum, horse serum, antibiotics, TNFα, human EGF and human bFGF was obtained from GIBCO. Nifedipine, methoxyverapamil hydrochloride (D-600), lanthanum chloride, Bapta-AM, caffeine, dantrolene, Stattic, Synta66, EGTA, BSA, cycloheximide and collagenase were obtained from Sigma-Merck; papain was obtained from Worthington Biochemicals. Dexamethasone, 2-APB, 5′-N-Ethylcarboxamidoadenosine (NECA), ATPγS, NNC55-0396, 78c (CD38 inhibitor), xestospongin C, FK506 and ryanodine were purchased from Tocris. Insulin and 8BrcADPr were obtained from Santa Cruz. Fura-2 AM was obtained from Invitrogen. All the lipophilic drugs were dissolved in dimethyl sulfoxide (ethanol in the case of xestospongin C). The final concentration of the solvent was ≤0.1%.
7
Generation of Murine Dendritic Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The generation of CD11c+ CD103+ CLEC9A+ DC was described previously.36 (link) Briefly, bone marrow was aseptically isolated from femurs and tibiae of untreated C57BL/6 mice and cultured in complete T cell medium for 16 days with the addition of 200 ng/mL rmFLT3L and 5 ng/mL rmGM-CSF (R&D Systems cat# 427-FL-005 and cat# 415 ML, respectively). Where indicated, after 10 days of initial culture with growth cytokines, AZD4635 at 3 µM, or DMSO as control was added and allowed to incubate for 30 min prior to addition of 5 µM 5′-(N-ethylcarboxamido) adenosine (NECA) (Tocris). The cultures were then continued with growth cytokines for the remaining 6 days prior to cell harvest. Immature dendritic cells (iDC) were induced to a mature phenotype with 1 µg/mL lipopolysaccharide (InvivoGen cat# tlrl-eblps) for 24 hours. Both iDC and mature DC were used for experiments where indicated. Before use in subsequent assays such as T cell co-cultures, DC were washed with Dulbecco’s PBS (DPBS), harvested with Accutase (Sigma cat# A6964) and then washed and resuspended in complete T cell media or flow cytometry staining buffer (ThermoFisher Scientific cat# 00-4222-26).
8
In Vitro and In Vivo Immune Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six- to eight-week-old female wild-type, OT-1 and OT-II mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal procedures were approved by the Institutional Animal Care and Use Committee of Merck & Co., Inc., Kenilworth, NJ, USA.
The pharmacologic HPK1 inhibitor Compound 1 was synthesized according to the methods described in Genentech patent application WO 2018183964 A1. Pembrolizumab and IgG4 isotype were generated by Merck & Co., Inc., Kenilworth, NJ, USA. lipopolysaccharide (LPS) (Sigma-Aldrich, St.Louis, MO), forskolin (FSK) (Sigma-Aldrich, St.Louis, MO), PGE2 (Cayman Chemical, Ann Arbor, MI), ovalbumin (OVA) peptides (OVA257-264 and OVA323-339) (Sigma-Aldrich, St.Louis, MO), and 5’-N-ethylcarboxamido adenosine (NECA) (Tocris, Bristol, UK) were prepared and used according to the manufacturer’s instructions. Anti-human CD3 (hCD3) (Clone OKT3), anti-human CD28 (hCD28) (Clone CD28.2), anti-mouse CD3 (mCD3) (Clone 145-2C11), anti-mouse CD28 (mCD28) (Clone 37.51), and carboxyfluorescein succinimidyl ester (CFSE) cell labeling kit were from Thermo Fisher Scientific (Waltham, MA). Mouse granulocyte macrophage-colony stimulating factor (GM-CSF) and mouse IL-4 were obtained from Peprotech (NJ, USA). All flow antibodies were purchased from Biolegend or BD Bioscience (San Diego, CA).
The pharmacologic HPK1 inhibitor Compound 1 was synthesized according to the methods described in Genentech patent application WO 2018183964 A1. Pembrolizumab and IgG4 isotype were generated by Merck & Co., Inc., Kenilworth, NJ, USA. lipopolysaccharide (LPS) (Sigma-Aldrich, St.Louis, MO), forskolin (FSK) (Sigma-Aldrich, St.Louis, MO), PGE2 (Cayman Chemical, Ann Arbor, MI), ovalbumin (OVA) peptides (OVA257-264 and OVA323-339) (Sigma-Aldrich, St.Louis, MO), and 5’-N-ethylcarboxamido adenosine (NECA) (Tocris, Bristol, UK) were prepared and used according to the manufacturer’s instructions. Anti-human CD3 (hCD3) (Clone OKT3), anti-human CD28 (hCD28) (Clone CD28.2), anti-mouse CD3 (mCD3) (Clone 145-2C11), anti-mouse CD28 (mCD28) (Clone 37.51), and carboxyfluorescein succinimidyl ester (CFSE) cell labeling kit were from Thermo Fisher Scientific (Waltham, MA). Mouse granulocyte macrophage-colony stimulating factor (GM-CSF) and mouse IL-4 were obtained from Peprotech (NJ, USA). All flow antibodies were purchased from Biolegend or BD Bioscience (San Diego, CA).
9
Adenosine Receptor Agonists and CYP4A Regulation
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!