Cl316 243

Manufactured by Bio-Techne

Sourced in United Kingdom, United States

CL316,243 is a synthetic compound developed by Bio-Techne. It is a selective β3-adrenergic receptor agonist, which is a class of molecules that can activate the β3-adrenergic receptor. The core function of CL316,243 is to serve as a research tool for studying the physiological roles and pharmacological properties of the β3-adrenergic receptor in various experimental models.

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Lab products found in correlation

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Close spelling variants of this product

CL316,243 disodium salt (5 citations) CL316,243 (CL; (4 citations)

46 protocols using cl316 243

Adipocyte Differentiation and Response to CL316,243

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For the culture of primary mice BA and WA, iBAT and sWAT were isolated from 3-week-old C57BL/6J male mice. The white preadipocytes were maintained in DMEM/F12 (Gibco–BRL Laboratories, Grand Island, NY, USA) supplemented with 2 mM l-glutamine, 100 U/mL penicillin (Sigma, St. Louis, MO, USA) and 10% fetal bovine serum (FBS) (Gibco–BRL Laboratories), while the brown preadipocytes were maintained in DMEM high glucose medium (Gibco–BRL Laboratories) supplemented with 5 mM HEPES, 100 U/mL penicillin (Sigma) and 20% FBS (Gibco–BRL Laboratories) both at 37 °C and 5% CO₂. To induce differentiation, postconfluent preadipocytes were cultured in a standard differentiation medium (the DMEM or DMEM/F12 contained 850 nM insulin and 1 nM triiodothyronine). For the first three days of differentiation period, 0.5 mM 3-isobutyl-1-methylxanthine, 0.125 mM indomethacin, 1 µM dexamethasone and 1 µM Rosiglitazone were also added. The cells were fully differentiated after 9 days of culture in the differentiation medium.
The cells were treated with CL316,243 (2 µM, Tocris Bioscience, Bristol, UK) for 1, 2, 3, 4, 5, 6, 12 and 24 h after the deprivation of FBS for 12 h. After CL316,243 treatment, mature adipocytes were lysed in Trizol for RNA extraction.

Luo X., Jia R., Zhang Q., Sun B, & Yan J. (2016). Cold-Induced Browning Dynamically Alters the Expression Profiles of Inflammatory Adipokines with Tissue Specificity in Mice. International Journal of Molecular Sciences, 17(5), 795.

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CL316243 Treatment Impacts Adipose Tissues

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For CL316243 treatment, fourteen six-week-old male C57BL/6J mice (purchased from Vital River Laboratory Animal Technology Co. Ltd.) were randomly divided into two groups, seven for each. One group was injected intraperitoneally (i.p.) once daily with 1 mg/kg CL316243 (Tocris Bioscience) in 0.9% NaCl for 7 days [33 (link)]; 0.9% NaCl was used in the control group instead of CL316243. The mice were maintained at 22 ± 2°C on a 12 h/12 h light cycle (8.00 a.m. to 8.00 p.m.) in Office of Laboratory Animal Welfare, certified animal facility, with free access to water and standard laboratory chow diet. Institutional Animal Care and Use Committee approved all experimental plans. Mice were killed by cervical dislocation. The interscapular brown adipose tissues, inguinal subcutaneous adipose tissues, and retroperitoneal epididymal adipose tissues were dissected out.

Zheng Z., Liu X., Zhao Q., Zhang L., Li C, & Xue Y. (2014). Regulation of UCP1 in the Browning of Epididymal Adipose Tissue by β3-Adrenergic Agonist: A Role for MicroRNAs. International Journal of Endocrinology, 2014, 530636.

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Antibody-based Adipose Tissue Characterization

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Antibodies to IRβ, P-T308-Akt, P-S473-Akt and Pan-Akt (all used at 1/1000 dilution) were from Cell Signaling. UCP1 antibody (#10983) was from AbCam (used at 1/100 for IHC and at 1/1000 for western blot). Live/dead Blue (Molecular Probes, L23105), CD31-PE-CY7 (eBioscience, 25-0311)(used at 1/1000), CD45-PE-CY7 (eBioscience, 25-0451) (used at 1/1000), CD29-AlexaFluor700 (Biolegend, 102218)(used at 1/400), CD34-AlexaFluor647 (Biolegend, 119314)(used at 1/200), Sca1-Pacific Blue (BD Bioscience, 560653)(used at 1/1000), CD45-FITC (eBioscience, 11-0451) (used at 1/200) were used for flow cytometry. CL316,243 was from Tocris.

Sanchez-Gurmaches J, & Guertin D.A. (2014). Adipocytes arise from multiple lineages that are heterogeneously and dynamically distributed. Nature communications, 5, 4099.

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Thermal Injury Model in C57BL/6 Mice

Cited 1 time

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Male C57BL/6 mice (Jackson Laboratory) were housed at ambient temperature and cared in accordance with the Guide for the Care and Use of Laboratory Animals. All procedures performed in this study were approved by the Sunnybrook Research Institute Animal Care Committee. Unless specified otherwise, full thickness (30% TBSA) was used to burn the mice. Full thickness was achieved in immersing the back of the mice (30% TBSA) at 98°C for 10 s, whereas partial thickness(30% TBSA)was achieved at 60°C for18s (Bayliss et al., 2014 (link)). Burned mice were subsequently housed individually in sterile cages and fed ad libitum until sacrifice. IL-6 knockout mice were obtained from Jackson Laboratories. CL316,243 (1 mg/kg/day) and propranolol (20 mg/kg/day) were obtained from Tocris Bioscience and administered i.p. immediately post-burn and twice daily (24 hr and 48 hr post-burn, respectively). Liposomal clodronate was purchased from Nico van Rooijen, PhD (Faculty of Medicine). They were prepared as previously described (Van Rooijen and Sanders, 1994 (link)). Mice were injected 48 hr prior to thermal injury and 48 hr post-thermal injury. Mice were sacrificed at 7 days post-thermal injury. Complete macrophages depletion was confirmed by immunostaining of Kupffer cells with CD14 (not shown).

Patsouris D., Qi P., Abdullahi A., Stanojcic M., Chen P., Parousis A., Amini-Nik S, & Jeschke M.G. (2015). Burn Induces Browning of the Subcutaneous White Adipose Tissue in Mice and Humans. Cell reports, 13(8), 1538-1544.

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Dissecting Metabolic Pathways through Pharmacological Modulation

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CL316243, forskolin, ⁸Br-cAMP, ARL 67156 trisodium salt, and suramin were procured from Tocris (Minneapolis, MN). Carbenoxolone, trovofloxacin, and spironolactone were from Sigma Aldrich (St. Louis, MO). Silencer select siRNAs against mouse Panx1 (assay ID-s79985) or mouse Gβ subunits (GNB1 – assay ID –ss66813; GNB2 – assay ID –ss66816; GNB3 – assay ID –ss66822; GNB4 – assay ID –n420113) and silencer select negative control-2 siRNA (4390846) were purchased from ThermoFisher Scientific (Middletown, VA). Antibodies used in the study were as follows: mouse monoclonal anti-Panx1 (Clone 720505, #MAB7097) was from R&D Systems (Minneapolis, MN); rabbit-polyclonal anti-UCP1 (#U6382) was from Sigma–Aldrich (St. Louis, MO); mouse monoclonal OXPHOS antibody cocktail (#MS604) was from Mitosciences (Eugene, OR); rabbit monoclonal anti-Panx1 antibody (Clone D9M1C, #91137), anti-PKC phosphorylation antibody sampler kit (#9921) and rabbit monoclonal anti-vinculin (Clone E1E9 V, #13901) were from Cell Signaling (Danver, MA). Cellular glucose uptake was measured using a glucose uptake-Glo assay kit (#J1341, Promega, Madison, WI).

Senthivinayagam S., Serbulea V., Upchurch C.M., Polanowska-Grabowska R., Mendu S.K., Sahu S., Jayaguru P., Aylor K.W., Chordia M.D., Steinberg L., Oberholtzer N., Uchiyama S., Inada N., Lorenz U.M., Harris T.E., Keller S.R., Meher A.K., Kadl A., Desai B.N., Kundu B.K, & Leitinger N. (2020). Adaptive thermogenesis in brown adipose tissue involves activation of pannexin-1 channels. Molecular Metabolism, 44, 101130.

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Adipose Tissue Lipolysis Assay

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For the in vivo lipolysis assay, blood was collected 0, 10, and 20 min after β3-adrenergic agonist CL316,243 (Tocris Bioscience) i.p. injection into mice. For ex vivo or in vitro lipolysis assays, 20 mg of epididymal, inguinal adipose tissue explants or differentiated 3T3-L1 cells were cultured in Krebs-ringer HEPES buffer (115 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 1.2 mM NaH₂PO₄, 2.5 mM CaCl₂, 25 mM NaHCO₃, 12 mM HEPES, pH 7.4) with 0.5% fatty acid-free BSA (Sigma) and 5 mM glucose. The medium was collected 0, 60, and 120 min after adding 1 μg/ml CL316,243, with levels of FFA and glycerol normalized to the weight of adipose explants or protein concentrations from 3T3-L1 cell lysates.

Kim K., Kang J.K., Jung Y.H., Lee S.B., Rametta R., Dongiovanni P., Valenti L, & Pajvani U.B. (2021). Adipocyte PHLPP2 inhibition prevents obesity-induced fatty liver. Nature Communications, 12, 1822.

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Pharmacological Induction and Measurement of Lipolysis in Mice

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Lipolysis was induced in 8- to 10-week-old mice using the pan-beta adrenergic agonist, ISO or the beta 3-specific agonist, CL-316,243. Mice were fasted beginning at 9 a.m. for 6 h, and lipolysis was induced by injection of freshly prepared and sterile-filtered ISO (10 mg/kg, IP, Tocris Bioscience, catalog no.: 1747) or CL-316,243 (0.1 mg/kg, IP, Tocris Bioscience, catalog no.: 1499) in PBS. About 70 μl of blood was collected from a tail nick into heparinized capillary tubes at baseline (0) before injection, and 15, 30, and 60 min after injection, for measurement of plasma FABP4, insulin, NEFA, corticosterone, and glycerol levels. For studies using hexamethonium (Hex), mice were injected IP with 20 mg/kg Hex (Tocris, catalog no.: 4111) or PBS 30 min prior to induction of lipolysis with CL-316,243. For thermoneutral experiments, mice were housed in the temperature-controlled chamber at 30°C throughout the lipolysis experiment, and blood was collected from mice under a heat lamp to prevent activation of thermogenic pathways. For FABP4 clearance studies, 10 μg of recombinant mouse FABP4 (produced in-house) was injected intraperitoneally into mice, and blood collected via tail vein at 10, 20, 60, and 120 min postinjection.

Prentice K.J., Lee A., Cedillo P., Inouye K.E., Ertunc M.E., Riveros J.K., Lee G.Y, & Hotamisligil G.S. (2023). Sympathetic tone dictates the impact of lipolysis on FABP4 secretion. Journal of Lipid Research, 64(6), 100386.

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Adrenergic Regulation of Hypothalamic eCB

Cited 1 time

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Hypothalamic eCB levels were determined after adrenergic stimulation of BAT thermogenesis with the selective β3-adrenergic agonist CL 316,243 (Tocris Bioscience, Bristol, UK) in lean mice, as previously described (26 (link), 27 (link)). At 4 h after ip injection of either CL 316,243 (10 mg/kg) or vehicle (aqueous buffer) (26 (link)), mice were euthanized by cervical dislocation, and hypothalamus and BAT were collected for further analysis.

Miralpeix C., Fosch A., Casas J., Baena M., Herrero L., Serra D., Rodríguez-Rodríguez R, & Casals N. (2019). Hypothalamic endocannabinoids inversely correlate with the development of diet-induced obesity in male and female mice. Journal of Lipid Research, 60(7), 1260-1269.

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Radiolabeled PET Tracers and Ligands Synthesis

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2-[¹⁸F]fluoro-2-deoxy-D-glucose ([¹⁸F]FDG) was prepared in-house using a fully automated cassette-based synthesizer (FASTlab, GE Healthcare, Uppsala, Sweden) within the clinical routine production at the Vienna General Hospital, Austria. [¹¹C]SNAP-7941 was synthesized as reported elsewhere using an automated module (TRACERlab FC X Pro, GE Healthcare, Uppsala, Sweden) (25 (link)). All PET-tracers were physiologically formulated and quality-controlled prior to administration. The adrenergic receptor beta ligands carazolol, pindolol and (S)-propranolol hydrochloride were purchased from Sigma-Aldrich (St. Louis, USA). The ADRB3 agonist CL 316,243 was purchased from Tocris Bioscience (Bristol, UK). The unlabeled compounds FE@SNAP and SNAP-7941 were synthesized at the Department of Pharmaceutical Chemistry and at the Department of Organic Chemistry (University of Vienna, Austria). The radioligands 5,7-[³H](-)CGP-12177 and [¹²⁵I](-)Iodocyanopindolol were purchased from PerkinElmer, Inc. (Waltham, USA). All other reagents and cell culture supplies were purchased from standard commercial sources.

Balber T., Benčurová K., Kiefer F.W., Kulterer O.C., Klebermass E.M., Egger G., Tran L., Wagner K.H., Viernstein H., Pallitsch K., Spreitzer H., Hacker M., Wadsak W., Mitterhauser M, & Philippe C. (2019). In vitro Radiopharmaceutical Evidence for MCHR1 Binding Sites in Murine Brown Adipocytes. Frontiers in Endocrinology, 10, 324.

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Mitochondrial Dynamics in Adipocyte Metabolism

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The following reagents were used: oligomycin, FCCP, rotenone, antimycin A, ADP, GDP, isoproterenol, CL 316,243 (Tocris Bioscience), isobutylmethylxanthine, dexamethasone, insulin, rosiglitazone, T3, recombinant mouse IL10 (Calbiochem, Cat# 407700), RNeasy (Qiagen), Lipofectamine 3000 (Life Technologies). DNA vectors: pDsRed2-Mito Vector (Clonetech). Antibodies as follows: anti-UCP-1 (Abcam, ab10983), anti- Phospho-(Ser/Thr) PKA Substrate (Cell Signaling #9621), anti-OPA1 (Abcam, ab42364), anti-mitofusin-2 (Abcam, ab 50843), anti-alpha-tubulin (Abcam, ab4074), anti- mtTFA ((A-17): sc-23588), anti-IL10 (Santa Cruz, sc-8438) and anti-OXPHOS cocktail (Abcam, ab110413).

de-Lima-Júnior J.C., Souza G.F., Moura-Assis A., Gaspar R.S., Gaspar J.M., Rocha A.L., Ferrucci D.L., Lima T.I., Victório S.C., Bonfante I.L., Cavaglieri C.R., Pareja J.C., Brunetto S.Q., Ramos C.D., Geloneze B., Mori M.A., Silveira L.R., Segundo G.R., Ropelle E.R, & Velloso L.A. (2018). Abnormal brown adipose tissue mitochondrial structure and function in IL10 deficiency. EBioMedicine, 39, 436-447.

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