Cck 8

Manufactured by Bachem

Sourced in United States, Switzerland

CCK-8 is a reagent used for the colorimetric determination of cell viability and cytotoxicity in cell proliferation and cytotoxicity assays. It utilizes a highly water-soluble tetrazolium salt that is reduced by living cells to produce a water-soluble formazan dye. The intensity of the color is directly proportional to the number of viable cells in the sample.

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Lab products found in correlation

Recombinant murine leptin, by Thermo Fisher Scientific (1 mentions) Arginine vasopressin, by Bachem (1 mentions) Cck 33, by Peptide Institute (1 mentions) Hek293 cell, by American Type Culture Collection (1 mentions) Cho k1 cells, by American Type Culture Collection (1 mentions) Exenatide, by Bachem (1 mentions) Ac187, by Bio-Techne (1 mentions) C18 reverse phase column, by Waters Corporation (1 mentions) 433a peptide synthesizer, by Thermo Fisher Scientific (1 mentions) C fos, by Cell Signaling Technology (1 mentions) Rabbit anti c fos primary antibody, by Cell Signaling Technology (1 mentions)

15 protocols using cck 8

CCK-SAP-Induced Ablation of CCK Receptors

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To validate CCK‐SAP‐induced ablation of CCK receptor expressing NG neurones, we tested the hypophagic responses to CCK in all rats from each cohort. Within subject design was used with each rat receiving counterbalanced vehicle (500 μL; saline) or sulphated CCK octapeptide (CCK‐8S, 4 μg/kg body weight, IP, Bachem, Torrance, CA, USA).⁵⁰ Animals were fasted for 16 hours on wire bottom cages and injected 1 hours into the dark cycle. The injections were administered in a counterbalanced fashion with half animals receiving the drug on first day followed by vehicle injection on second day, and others receiving vehicle on first day followed by the drug injection on second day. Food weight and spillage were manually recorded for 2 hours or in an automated episodic food intake monitoring system (BioDAQ). Rats with greater than 25% reduction in food intake in response to CCK (4 μg/kg; IP) compared to saline were included in the SAP group, and rats with less than 5% satiation after CCK compared to saline were included in CCK‐SAP group. Three rats were excluded based on these parameters.

McDougle M., Quinn D., Diepenbroek C., Singh A., de la Serre C, & de Lartigue G. (2020). Intact vagal gut‐brain signalling prevents hyperphagia and excessive weight gain in response to high‐fat high‐sugar diet. Acta Physiologica (Oxford, England), 231(3), e13530.

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CCK-Induced Satiety in Rats

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Rats were deprived of food during 15 h and were intraperitoneally injected with either sterile NaCl 0.9% (saline) or CCK octapeptide, sulfated (CCK-8S, Bachem, Germany) just before light off and refeeding. FI and spillage were weighed every 30 min during 90 min. The satiating effect of CCK-8S was tested on distinct animals, for three doses [0.25, 2.5, and 7.5 nmol/kg of bodyweight (BW)] vs vehicle (saline) on consecutive days. It means that each rat received one of the treatment on a given test day followed by a period of 3–5 days of wash out and resting between each injection. Therefore, the whole experiment extended over 3 weeks. Doses of CCK were chosen on the basis of previous published data. The low dose (0.25 nmol/kg BW) reduces 1-h FI by 25% in standard fed rats (28 (link)). The 10-fold higher dose (2.5 nmol/kg BW) is necessary to induce FI reduction in diet-induced obesity rats (29 (link)) and the higher dose (7.5 nmol/kg BW) is used to induce anorectic effect in MC4R^−/− obese rats (30 (link)).

Ndjim M., Poinsignon C., Parnet P, & Le Dréan G. (2017). Loss of Vagal Sensitivity to Cholecystokinin in Rats Born with Intrauterine Growth Retardation and Consequence on Food Intake. Frontiers in Endocrinology, 8, 65.

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Pharmacological Modulation of Metabolism

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CNO (1 mg/kg, Sigma), CCK-8s (2-10 μg/kg, Bachem), recombinant murine leptin (2 μg/kg, PeproTech), and Ex4 (3-30 μg/kg, American Peptide) were dissolved in sterile 0.9% saline. All drugs were administered i.p. (10 μl per gram body weight).

Campos C.A., Bowen A.J., Schwartz M.W, & Palmiter R.D. (2016). Parabrachial CGRP Neurons Control Meal Termination. Cell metabolism, 23(5), 811-820.

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Fasting Mice, CCK8s Injection, Food Intake

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After 5 weeks of treatment, animals were fasted for 12 hrs on wire bottom cages and injected 1 hr into the dark cycle with either CCK8s (0.3 μg/kg, IP, Bachem, Torrance, CA, USA) or saline (400μl; vehicle). Food intake was recorded for 2 hrs following injection.

de La Serre C.B., de Lartigue G, & Raybould H.E. (2014). Chronic exposure to Low dose bacterial lipopolysaccharide inhibits leptin signaling in vagal afferent neurons. Physiology & behavior, 139, 188-194.

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Validating CCK-SAP Induced Ablation

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To validate CCK-SAP induced ablation of CCK receptor expressing NG neurons we tested the hypophagic responses to CCK in all rats from each cohort. Within subject design was used with each rat received counterbalanced vehicle (500μl; saline) or sulfated CCK octapeptide (CCK-8S, 4 μg/kg body weight, IP, Bachem, Torrance, CA, USA)⁵⁰. Animals were fasted for 16 hrs on wire bottom cages and injected 1 hr into the dark cycle. The injections were administered in a counterbalanced fashion with half animals receiving the drug on first day followed by vehicle injection on second day, and others receiving vehicle on first day followed by the drug injection on second day. Food weight and spillage were manually recorded for 2h or in an automated episodic food intake monitoring system (BioDAQ). Rats with greater than 25% reduction in food intake in response to CCK (4 μg/kg; IP) compared to saline were included in the SAP group, and rats with less than 5% satiation after CCK compared to saline were included in CCK-SAP group. Three rats were excluded based on these parameters.

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Vagotomy Confirmation in Feeding Studies

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The importance of acclimatization to the experimental procedures in the feeding response to GI hormones has been previously documented [22 (link)]. Therefore, rats were handled on a daily basis from 5 days post-surgery and familiarized to the experimental procedures. To confirm that the vagus nerve ablation was successful, a procedure based on the feeding response to cholecystokinin octapeptide (CCK-8) (10 µg/kg ip) (Bachem, St Helens, UK) was used as previously described [2 (link),21 (link)]. Further confirmation of vagotomy success or failure was obtained by weighing the rats’ stomachs after the sacrifice. As previously described [23 (link)], a 2-fold increase in wet stomach size can be considered a marker of a successful vagotomy.
Rats in the vagotomized group that exhibited an anorectic response to CCK-8 (inhibition of food intake of 50% or more) when compared to the saline control and with a stomach weight less than 2-fold greater as compared to the average stomach weight of sham controls were excluded from analysis (16.6% vagotomized rats were excluded).

Morais T., Pereira S.S., Andrade S., Neves D., Guimarães M., Nora M., Carreira M.C., Casanueva F.F, & Monteiro M.P. (2023). GLP-1 Increases Circulating Leptin Levels in Truncal Vagotomized Rats. Biomedicines, 11(5), 1322.

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CCK-8 Assay for Pancreatic Cell Function

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CCK-8 was purchased from Bachem, Bubendorf, Switzerland; L364718 from Merck, Darmstadt, Germany; collagenase (CLSPA grade) from Worthington, NJ, USA; amylase detection reagent from Boehringer Mannheim, Germany; EU-RIA CCK kit for CCK measurements from EuroDiagnostic, Malmö, Sweden; Sephadex G-50 from Pharmacia, Freiburg, Germany; and C18 Sep-Pac cartridges were from Water Corporation, Milford, MA, USA. All other reagents were purchased from Sigma, Deisenhofen, Germany.

Egberts J.H., Raza G.S., Wilgus C., Teyssen S., Kiehne K, & Herzig K.H. (2020). Release of Cholecystokinin from Rat Intestinal Mucosal Cells and the Enteroendocrine Cell Line STC-1 in Response to Maleic and Succinic Acid, Fermentation Products of Alcoholic Beverages. International Journal of Molecular Sciences, 21(2), 589.

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Synthesis and Characterization of CCK Analogues

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CCK peptide analogues were custom synthesized in our laboratory, purified to homogeneity, and verified by mass spectrometry [7 (link)]. These include natural CCK-26-33 (CCK-8); a partial agonist analogue, D-Tyr-Gly-[(Nle^28,31)CCK-26-32]-phenethyl ester (CCK-OPE); and a fluorescent analogue of this hormone, alexa488-D-Tyr-Gly-[(Nle^28,31)CCK-26-33] (alexa488-CCK) [8 (link)]. The sulfated CCK octapeptide (CCK-8) (#4033010) used in the structure–activity relationship studies and arginine vasopressin (#4012215) were purchased from Bachem AG (Bubendorf, Switzerland). CCK-33 was purchased from Peptides International (Louisville, KY, USA). Clonal receptor-bearing cell lines were prepared from non-CCK receptor-bearing CHO-K1 cells or HEK-293 cells (American Type Culture Collection, ATCC), as previously described [9 (link)]. In select experiments, the cholesterol composition of cell lines was enhanced by treatment with methyl-β-cyclodextrin-cholesterol inclusion complex, as we previously described [10 (link)].

Dengler D.G., Harikumar K.G., Yen A., Sergienko E.A, & Miller L.J. (2023). Mechanism of Action and Structure–Activity Relationships of Tetracyclic Small Molecules Acting as Universal Positive Allosteric Modulators of the Cholecystokinin Receptor. Membranes, 13(2), 150.

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Vagal Afferent Deafferentation in Rats

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SDA vagotomy involves left-side intracranial vagal rhizotomy for afferent rootlet resection and transection of the dorsal subdiaphragmatic trunk of the vagus nerve along the esophagus in rats anesthetized with ketamine/xylazineas previously described Klarer et al., 2014 (link)). Rats received liquid diet during the initial 3 d and were offered wet chow before returning to solid diet. The recovery period extended for two wk. To test the success of the deafferentation, rats were food deprived overnight and injected intraperitoneally (ip) with 4 μg/kg CCK-8 (Bachem) or vehicle (PBS) 2 h after the onset of the light phase. Food was returned and intake was monitored after 1 and 2 h. Any CCK-treated SDA rat eating < 3.4 g after 2 h (average 6.5 g of the rats left in the experiment) was removed from the study.

Holland J., Sorrell J., Yates E., Smith K., Arbabi S., Arnold M., Rivir M., Morano R., Chen J., Zhang X., Dimarchi R., Woods S.C., Sanchez-Gurmaches J., Wohleb E, & Perez-Tilve D. (2019). A Brain-Melanocortin-Vagus Axis Mediates Adipose Tissue Expansion Independently of Energy Intake. Cell reports, 27(8), 2399-2410.e6.

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Exenatide and CCK-8 Analogues Protocol

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Exenatide and CCK-8 were purchased from Bachem. AC3174 (an Exenatide analogue), AC170222 (a CCKR1-selective agonist), and AC170236 (a CCKR2-selective agonist) were synthesised at Amylin Pharmaceuticals and provided via MedImmune/AstraZeneca through its acquisition of Amylin Pharmaceuticals as previously described [10 ]. AC187 was purchased from Tocris and used at 100 μg/kg.

Roth E., Benoit S., Quentin B., Lam B., Will S., Ma M., Heeley N., Darwish T., Shrestha Y., Gribble F., Reimann F., Pshenichnaya I., Yeo G., Baker D.J., Trevaskis J.L, & Blouet C. (2020). Behavioural and neurochemical mechanisms underpinning the feeding-suppressive effect of GLP-1/CCK combinatorial therapy. Molecular Metabolism, 43, 101118.

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