Rin5F cells were purchased from ATCC (Cat. # CRL-2058). 90 μL of cells were plated at 60,000/ml cells, in 96-well plates (Cat. # 3603, Costar, Fisher Scientific) in DMEM media (Life Technologies, cat. 11965092) supplemented with 10% (vol/vol) FBS (Life Technologies, cat. A3160401), 1% penicillin/streptomycin (Life Technologies, cat. 15140122), and 1% Glutamax (Life Technologies, cat. 35050061) at 37 °C, 5% CO2 in a humidified incubator. To perform MTT assays, patient-fibril-seeded hIAPP fibrils in 50 μM of concentration were pelleted by centrifugation at 21,000 × g for 1 hour and supernatant was removed. The pellet was suspended in sterile PBS buffer and sonicated for 5 minutes, and 10 μL of sample solution was added to cells at various concentrations of 0, 1, 10 and 50 μM. Experiments were done in triplicate. After a 24-hour incubation of samples with cells, 20 μL of Thiazolyl Blue Tetrazolium Bromide MTT dye (Sigma) was added to each well and incubated for 3.5 h at 37°C under sterile conditions. The MTT dye stock was prepared by dissolving 5 mg/mL in sterile PBS buffer. The MTT assay was stopped by carefully aspirating off the culture media and adding 100 μL of 100% DMSO to each well. Absorbance was measured at 570 nm and the background reading was recorded at 700 nm and subsequently subtracted from the 570 nm value.
Rin 5f cells
Manufactured by American Type Culture Collection
Sourced in United States
Rin-5F cells are a type of insulin-secreting cell line derived from a rat insulinoma. They are used in research to study insulin production and pancreatic beta cell function.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Thiazolyl blue tetrazolium bromide mtt dye, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
Ins 1 cells, by Addexbio (1 mentions)
Sodium succinate, by Merck Group (1 mentions)
Resorufin, by Merck Group (1 mentions)
Methoxyresorufin, by Merck Group (1 mentions)
Cumene hydroperoxide, by Merck Group (1 mentions)
Dodecyl maltoside, by Merck Group (1 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Thermo Fisher Scientific (1 mentions)
Glutathione reductase, by Merck Group (1 mentions)
1 chloro 2 4 dinitrobenzene, by Merck Group (1 mentions)
7 ethoxyresorufin, by Merck Group (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Coenzyme q2, by Merck Group (1 mentions)
Glut2, by Santa Cruz Biotechnology (1 mentions)
Atp bioluminescent somatic cell assay kit, by Merck Group (1 mentions)
Apoptosis detection kits for flow cytometry, by BD (1 mentions)
5 protocols using rin 5f cells
1
MTT Assay for Fibril Cytotoxicity
2
Culturing Diverse Cell Lines for Experimentation
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Cell lines were cultured in RPMI-1640 with 10% fetal bovine serum (Invitrogen), 2 mmol/L glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin. INS-1 cell media was additionally supplemented with 50 umol/L 2-mercaptoethanol. HeLa and RIN-5F cells were obtained from ATCC. INS-1 cells were obtained from AddexBio. The firefly luciferase-expressing HeLa cell line were derived from a stable line used previously in our lab [58 ].
3
Streptozotocin-Induced Diabetes Assay
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Streptozotocin (STZ), reduced and oxidized glutathione (GSH/GSSG), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), NADH, NADPH, cytochrome c, coenzyme Q2, sodium succinate, antimycin A, dodecyl maltoside, resorufin, 7-ethoxyresorufin, methoxyresorufin, Hoechst 33342, and ATP bioluminescent somatic cell assay kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2′,7′-Dichlorofluorescein diacetate (DCFDA) was procured from molecular probes (Eugene, OR, USA). Kits for nitric oxide and caspase-3 and caspase-9 assays were purchased from R&D Systems Inc., MN, USA, and that for lipid peroxidation (LPO) from Oxis International Inc. (CA, USA). Kits for GSH/GSSG assay were procured from Promega Corp. (Madison, WI, USA). Apoptosis detection kits for flow cytometry were purchased from BD Pharmingen (BD Biosciences, San Jose, USA). Rin-5F cells were obtained from American Type Culture Collection (Manassas, VA, USA). Polyclonal antibodies against beta-actin, caspase-3, PARP, NOS-2, Nrf2, GLUT 2, Bax, Bcl-2, Akt, and p-Akt were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Reagents for cell culture, SDS-PAGE, and Western blot analyses were purchased from Gibco BRL (Grand Island, NY, USA) and Bio-Rad Laboratories (Richmond, CA, USA).
4
Peptide-induced cAMP Measurement in Rin-5F cells
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Activity was assayed based on the peptide-induced increase in extracellular cAMP using a Rin-5F rat pancreas  cell line [9, 37] . Rin5F cells, obtained from the American Type Culture Collection (ATCC), were grown in 12 well plates as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 units/mL penicillin and 100 g/mL streptomycin at 37 • C in a 5% CO 2 atmosphere. After 4 h of serum free culturing cells were challenged for 10 min with 100 L of 100 nM of the GLP-1 analogs in triplicate. The cAMP in the medium was measured using the cAMP EIA kit (Enzo Life Science) according to the manufacturer's protocol using synthetic human GLP-1-(7-36)NH 2 and exendin-4-(1-39)NH 2 [6] , a lactam(30-34) GLP-1 analog, LP1-19, and the GLP-1 receptor antagonist exendin-9 [10] for control experiments. Activities were measured with and without the antagonist exendin-9-(9-36)NH 2 , which was added 10 min prior to the agonist GLP-1 analogs. For dose response curves a Dis-coverX cAMP assay was applied. Peptides were dissolved in DPBS buffer, purchased from Biowest, with 0.1% BSA.
5
Cell Culture and Differentiation Protocol
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RIN-5F cells derived from rat insulinoma and 3T3-L1 preadipocytes derived from 3T3 mouse embryo fibroblast were purchased from the American Type Culture Collection (Manassas, VA; ATCC no.: CRL-2058 and CL-173, resp.). The RIN-5F cells were cultured in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin under an atmosphere of 5% CO2/95% humidified air at 37°C. The medium was renewed every 3 days. The cells were used at passages 20–25. The 3T3-L1 preadipocytes were grown and differentiated into adipocytes as described previously [46 (link)]. Briefly, preadipocytes were differentiated in high-glucose DMEM, 10% FBS with dexamethasone (0.25 μM), insulin (10 μg/mL), and 3-isobutyl-1-methylxanthine (0.5 mM) for 48 h and then treated with insulin (1 μg/mL) for an additional 48 h. Adipocytes were maintained in and refed every 2 days with high-glucose DMEM and 10% FBS until being used for experiments 8–12 days after the addition of differentiation factors, when between 90 and 95% of cells exhibited an adipocyte phenotype. For antidiabetic drugs or herbal ingredient treatment of cells, RIN-5F and 3T3-L1 adipocytes were cultured for the indicated time in the corresponding medium containing BSA (2%) and glucose (5.4 mM) in the absence of nateglinide, pioglitazone, berberine, and 4-Hydroxymephenytoin. After that, other assays were performed.
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