Co2 incubator

Manufactured by Binder

Sourced in Germany, United States

The CO2 incubator is a laboratory equipment designed to maintain a controlled, temperature-regulated and CO2-enriched environment, suitable for cell culture applications. It ensures consistent and stable conditions to support the growth and proliferation of cells.

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36 protocols using co2 incubator

Cytotoxicity Evaluation of Dox Compositions

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The cytotoxicity of the developed Dox compositions was evaluated in vitro using the MTT test. HT-1080 cells (10⁵ cells per well) were seeded into sterile 96-well culture plates and incubated at 37 °C in 5% CO₂ using a Binder CO₂ incubator (Binder, Tuttlingen, Germany) for 24–26 h. Then, the test substances were added at Dox concentrations of 25, 10, 5, 1, 0.1, and 0 µM and the cells were incubated for 24 h and 48 h.
After that, 20 µL of MTT (5 mg/mL) was carefully added to each well and incubated at 37 °C for 4 h. The medium was then carefully removed and 150 µL of MTT solvent was added. The cells were covered with foil and shaken on an orbital shaker for 15 min. The absorbance was recorded at 550 nm using Multiscan FC (ThermoSpectronic, Waltham, ML, USA) and normalized to the untreated control (without Dox).

Kostryukova L.V., Tereshkina Y.A., Tikhonova E.G., Khudoklinova Y.Y., Bobrova D.V., Gisina A.M., Morozevich G.E., Pronina V.V., Bulko T.V, & Shumyantseva V.V. (2023). Effect of an NGR Peptide on the Efficacy of the Doxorubicin Phospholipid Delivery System. Nanomaterials, 13(15), 2229.

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Culturing Human Fibrosarcoma Cells

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The study used a human fibrosarcoma cell line (HT-1080 cell culture) obtained at the D. I. Ivanovskiy Institute of Virology (Moscow, Russia). Tumor cells were cultured according to the recommendations in the cell culture certificate using appropriate media fortified with 10% FBS [25 (link)]. The cells were cultured at 37 °C in a humid atmosphere with 5% CO₂ (Binder CO₂ Incubator, Binder, Tuttlingen, Germany). Cell lines were used after 3 to 10 passages.

Tereshkina Y.A., Kostryukova L.V., Tikhonova E.G., Khudoklinova Y.Y., Orlova N.A., Gisina A.M., Morozevich G.E., Melnikov P.A, & Pokrovsky V.S. (2022). Chlorin e6 Phospholipid Delivery System Featuring APN/CD13 Targeting Peptides: Cell Death Pathways, Cell Localization, In Vivo Biodistribution. Pharmaceutics, 14(10), 2224.

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HDF cell culture conditions

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HDF were obtained from CellnTec (Bern, Switzerland). Cells were grown in Dulbecco’s Modified Eagle Medium: nutrient mixture F-12 (DMEM/F-12, Thermo Fischer Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Sigma-Aldrich, Lot: 065M3352), 1% penicilin–streptomyocin (P/S, Sigma-Aldrich, Buchs, Switzerland), and maintained in a humidified 5% CO₂ incubator (Binder GmbH, Tuttlingen, Germany) at 37 °C. The cells were kept at a confluence <80% prior to the experiments, and cell passages of p = 9 and p = 12 were used for the short-term and the long-term study, respectively.

Merk M., Chirikian O, & Adlhart C. (2021). 3D PCL/Gelatin/Genipin Nanofiber Sponge as Scaffold for Regenerative Medicine. Materials, 14(8), 2006.

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Preparation and Use of Glycyrrhetinic Acid Solution

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The human bladder cancer T24 cell line was obtained from China Center for Type Culture Collection (CCTCC, No : GDC078). T24 cells were cultured in MEM medium (Gibco) with 10% FBS, 100 units/ml penicillin and 100 units/ml streptomycin, then maintained in 37°C with 5% CO₂ incubator (Binder, Germany). Preparing GA stock solution at a concentration of 500 µg/ml: precisely weighed 2.00 mg GA in a EP tube, added 4 ml FBS free MEM medium, mixed with a vortex to ensure complete dissolution, and then stored at -20°C. The GA stock solution needs to be diluted before use. Specifically, the GA stock solution was taken out and dissolved in the dark at 37°C, and then it was diluted to the appointed concentration with FBS-free MEM medium to obtain the working solution. Attention should be paid here to avoid light in the use of GA.

Zeng M., Su Y., Li K., Jin D., Li Q., Li Y, & Zhou B. (2020). Gallic Acid Inhibits Bladder Cancer T24 Cell Progression Through Mitochondrial Dysfunction and PI3K/Akt/NF-κB Signaling Suppression. Frontiers in Pharmacology, 11, 1222.

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In vitro Examination of PC and HEK Cells

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Human PC cell line DU145 along with the human embryonic kidneys (HEK293T) which were purchased from the National Institute of Genetic Engineering and Biotechnology cell bank (NIGEB Tehran, Iran) were considered for in vitro examinations. We used Roswell Park Memorial Institute (RPMI1640, Invitrogen, USA) and Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) for culturing DU145 and HEK293T cells, respectively. Each media was supplemented with 100,000 U/L of penicillin, 100 μg/ml streptomycin (Fluka, Switzerland), and 10% fetal bovine serum (FBS, Biowest, France). Cells incubation was carried out in a humidified 5% CO2 incubator at 37 °C (Binder, USA).

Taghizadeh S., Soheili Z.S., Sadeghi M., Samiei S., Ranaei Pirmardan E., Kashanian A., Zakeri F., Latifi-Navid H, & Shams Najafabadi H. (2021). sFLT01 modulates invasion and metastasis in prostate cancer DU145 cells by inhibition of VEGF/GRP78/MMP2&9 axis. BMC Molecular and Cell Biology, 22, 30.

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Early Peptide Toxicity Screening

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To determine early signs of peptide toxicity, the library was tested against the MRC5-SV2 cell line of human, embryonic lung fibroblasts (Sigma-Aldrich). Peptides were serially diluted in 96-well plates as described earlier at concentrations ranging from 64 μM to 0.25 μM for the first screening, and from 32 μM to 0.016 μM for the independent repeat. Tamoxifen (Sigma-Aldrich) was included as a reference compound. After preparation of the test plates, cell suspension (190 μL) of 1.5 × 10^5 cells/mL in complete minimum essential medium (Gibco) was added to the peptides. Plates were incubated for 72 h at 37°C in a 5% CO₂ incubator (Binder). Afterwards, 50 μL of a 0.01% (wt/vol) resazurin (Sigma-Aldrich) solution was added to the wells to detect cell viability. After 4 h of incubation, fluorescence was read using a microplate reader (Telix) at λ_excitation = 550 nm and λ_emission = 590 and the IC₅₀ values were calculated.

Van Moll L., De Smet J., Paas A., Tegtmeier D., Vilcinskas A., Cos P, & Van Campenhout L. (2022). In Vitro Evaluation of Antimicrobial Peptides from the Black Soldier Fly (Hermetia Illucens) against a Selection of Human Pathogens. Microbiology Spectrum, 10(1), e01664-21.

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Cell Culture Maintenance Protocol

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The Caco-2 cell line was cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine, while the HepG-2 and MCF-7 cell lines were maintained in RPMI-1640 augmented with 10% FBS and 2 mM L-glutamine. All the mentioned cell lines were incubated at 37 °C, in a humidified 5% CO₂ incubator (BINDER, Germany). The cells were passaged twice to thrice per week, enzymatically with 0.25% trypsin-EDTA (1 X) and subcultured in 25 cm² tissue culture flasks.

El-Baky N.A., EL-Fakharany E.M., Sabry S.A., El-Helow E.R., Redwan E.M, & Sabry A. (2022). A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha. Biology, 11(2), 157.

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Oxygen and Glucose Deprivation Model

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Oxygen and glucose deprivation (OGD): when grown to confluence, pBVECs were cultured in FBS-, Sodium Pyruvate- and Glucose-free F12/DMEM culture medium (Gibco) in a 0.2% O₂ and 5% CO₂ incubator (Binder) at 37˚C for 20 hours. Low oxygen and nutrition (LON): after damaged by OGD, ECs were cultured in hypoxia (2% O₂) and inadequate nutrition culture medium for 24 hours (F12/DMEM medium containing 20% FBS was diluted to four times of volume with deionized water). For pathway study, cells were pre-treated with PD98059 (10 μmol/ml), Rapamycin (0.1 μmol/ml) and LY249002 (10 μmol/ml) for 1 hour to inhibit ERK, mTOR, and AKT respectively. And then, cell apoptosis and necrosis in the model of OGD+LON were detected.

Liu Y., Tang Q., Shao S., Chen Y., Chen W, & Xu X. (2017). Lyophilized Powder of Catalpol and Puerarin Protected Cerebral Vessels from Ischemia by Its Anti-apoptosis on Endothelial Cells. International Journal of Biological Sciences, 13(3), 327-338.

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Antiproliferative Effect of Fe3O4 MNPs on Cancer Cells

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The antiproliferative effect of Fe₃O₄ MNPs on various cancer cell lines and a normal Chang liver cell line was quantified using a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) kit (Sigma-Aldrich) according to the standard method.28 (link) Briefly, cells were allowed to grow in a 75 cm² cell culture flask (TPP Techno Plastic Products, Trasadingen, Switzerland) until 95% confluent. The cells were then seeded into each well of a 96-well microculture plate (TPP Techno Plastic Products) at a concentration of 1×10⁵ cells/mL and treated with Fe₃O₄ MNPs at various concentrations. After incubation for 72 hours at 37°C in a 5% CO₂ incubator (Binder GmbH), 25 μL of a 5.5 mg/mL MTT solution was added to each well, covered with aluminum foil, and incubated for a further 3 hours in the dark. Immediately afterwards, the medium was aspirated and the remaining purple formazan was lysed with MTT solution. The assay was performed in triplicate. The optical density was then measured at 570 nm using an enzyme-linked immunosorbent assay universal microplate reader (Bio Tek Instruments, Inc., VT, USA). The inhibitory concentration 50 (IC₅₀) value was determined from the absorbance versus concentration curve. Dimethyl sulfoxide 0.1% was used as the negative control.

Namvar F., Rahman H.S., Mohamad R., Baharara J., Mahdavi M., Amini E., Chartrand M.S, & Yeap S.K. (2014). Cytotoxic effect of magnetic iron oxide nanoparticles synthesized via seaweed aqueous extract. International Journal of Nanomedicine, 9, 2479-2488.

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MTT Assay for SW620 Cell Proliferation

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SW620 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were maintained in Leibovitz’s L-15 Medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco) and 1% antibiotic–antimycotic (Gibco) at 37 °C in a 5% CO₂ incubator (BINDER GmbH, Tuttlingen, Germany). For MTT assay, the cell proliferation kit I (Roche Diagnostics, Basel, Switzerland) was used according to the manufacturer’s instructions. Briefly, SW620 cells were seeded in a 96-well plate at 1 × 10⁴ cells/well and incubated at 37 °C in a CO₂ incubator. After 24 h of incubation, the cells were treated with COS, GAand COS–GA at different concentrations (62.5, 122, and 250 µg/mL) for 24 and 48 h. MTT solution (10 µL) was added to each well, and the cells were continuously incubated for 4 h in a CO₂ incubator before adding 100 µL of solubilization solution. After 16–18 h of incubation, the absorbance of each well was measured at 570 nm using a FLUOstar^® Omega microplate reader (BMG Labtech, Ortenberg, Germany).

Saetang J., Sukkapat P., Mittal A., Julamanee J., Khopanlert W., Maneechai K., Nazeer R.A., Sangkhathat S, & Benjakul S. (2023). Proteome Analysis of the Antiproliferative Activity of the Novel Chitooligosaccharide–Gallic Acid Conjugate against the SW620 Colon Cancer Cell Line. Biomedicines, 11(6), 1683.

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