Its premix universal culture supplement

Manufactured by Corning

Sourced in United States

ITS+ Premix Universal Culture Supplement is a sterile, ready-to-use solution designed to support the growth and maintenance of a wide range of cell types in cell culture applications. The supplement contains insulin, transferrin, and selenium, which are essential components for cell growth and proliferation.

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10 protocols using its premix universal culture supplement

IPEC-J2 Cell Culture and Infection

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Nontransformed porcine jejunal epithelial cells (IPEC-J2) were grown in co-culture media that included Dulbecco’s minimal essential medium:nutrient mixture F-12 (with L-glutamine and 15 mmol/L HEPES, catalog 10-092-CM; Corning, Corning, NY) supplemented with 5 μg/mL each of insulin, transferrin, and selenium (ITS Premix universal culture supplement, catalog 354350; Corning), epidermal growth factor (5 ng/mL, catalog 354052; Corning), penicillin (50,000 IU/mL), streptomycin (50,000 mg/mL) (100× penicillin-streptomycin solution, catalog 30002CI; Corning), and 5% porcine serum (catalog 26250084; Thermo Fischer, Waltham, MA), and incubated at 37°C in 5% CO₂. Cells were seeded onto permeable polycarbonate filters (0.4-μm pore size, either 0.6 cm² or 4.67cm²; catalog PIHP01250 and PIHP03050, respectively; Millipore Sigma, Burlington, MA) and cultured until confluent (TEER, ≥2000 Ω × 0.6 cm² or ≥1300 Ω × 4.67 cm²). TEER was measured using an EVOM2 epithelial voltohmmeter with chopstick electrodes (World Precision Instruments, Sarasota, FL). For immunofluorescence assessment of C parvum burden, IPEC-J2 cells were seeded onto 8-well chamber slides (Nunc Lab-Tek II, catalog 154534; Thermo Fisher, Waltham, MA) and grown to confluence over a period of 3–4 days before use. IPEC-J2 cells were used at passages 38–50. Media was changed every 3–4 days.

Ferguson S.H., Foster D.M., Sherry B., Magness S.T., Nielsen D.M, & Gookin J.L. (2019). Interferon-λ3 Promotes Epithelial Defense and Barrier Function Against Cryptosporidium parvum Infection. Cellular and Molecular Gastroenterology and Hepatology, 8(1), 1-20.

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Glioma and Adrenal Cell Line Protocols

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The human glioma cell line MGM-1 was a gift from Dr Hiroaki Kataoka (University of Miyazaki). MGM-1 cells were grown in Dulbecco’s modified Eagle medium (Gibco, Thermo Fisher Scientific #11965092) with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich #12306C) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Thermo Fisher Scientific #15140122) at 37 °C and 5% CO₂. The adrenal cortical carcinoma cell line NCI-H295R (referred to as H295R-S1; #CRL-2128) was purchased from the American Type Culture Collection and grown in Dulbecco’s modified Eagle medium/F-12, GlutaMAX with 2.5% Nu-Serum (Corning, #355100), 100 IU/ml penicillin, 100 μg/ml streptomycin, plus ITS+ Premix Universal Culture Supplement at a concentration recommended by the manufacturer (Corning, #354352). Cells were passaged using trypsin/EDTA 0.25% (Gibco, Thermo Fisher Scientific #25200056), and a maximum of 10 passages were used for all cell lines. For collection of cell pellets for CYP450 screening and antibody testing, MGM-1, MGM-3, NHA, HMC3, MA-10, and Huh7 cells were cultured and pelleted according to our previously published methods (22 , 66 ).

Lin Y.C., Cheung G., Zhang Z, & Papadopoulos V. (2023). Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells. The Journal of Biological Chemistry, 299(8), 105035.

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Culturing A549 and NCI-H441 Lung Cancer Cells

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The human alveolar carcinoma cell line A549 (ATCC, Manassas, VA, USA) was cultured at 37 °C/5% CO2 in RPMI supplemented 10% FCS and penicillin/streptomycin (100 U/mL; 100 µg/mL) in uncoated T25 flasks. The human lung adenocarcinoma cell line NCI-H441 (ATCC) were cultured in DMEM supplemented with Corning^® Insulin/Transferrin/Selenium (ITS) Premix Universal Culture Supplement (Corning, Bedford, MA, USA) 10% FCS and penicillin/streptomycin (100 U/mL; 100 µg/mL) in uncoated T25 flasks. Cells were passaged at ~90% confluence using trypsin-EDTA.

Kruk D.M., Wisman M., Noordhoek J.A., Nizamoglu M., Jonker M.R., de Bruin H.G., Arevalo Gomez K., ten Hacken N.H., Pouwels S.D, & Heijink I.H. (2021). Paracrine Regulation of Alveolar Epithelial Damage and Repair Responses by Human Lung-Resident Mesenchymal Stromal Cells. Cells, 10(11), 2860.

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Cultivating H295R Adrenocortical Cells

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NCl-H295R (H295R) cells were purchased through American Type Culture collection (ATCC CRL-2128; Manassas, VA). H295R cells were grown at 37°C and 5% CO₂ in DMEM/F12 medium (Gibco Invitrogen, Carlsbad, CA) containing 2.5% FBS (HyClone, South Logan, UT), 2 mM L-glutamine (BioWhittaker Cambrex, East Rutherford, NJ), 1% ABAM (A5955, Sigma-Aldrich) and 1% ITS+ Premix Universal Culture Supplement (354352, Corning, Cleveland, TN). For analysis of response to atrazine and Ang II, cells were sub-cultured onto 12-well dishes at a density of 1.5 × 10⁵ cells/well.

Zimmerman A.D., Mackay L., Kemppainen R.J., Jones M.A., Read C.C., Schwartz D, & Foradori C.D. (2021). The Herbicide Atrazine Potentiates Angiotensin II-Induced Aldosterone Synthesis and Release From Adrenal Cells. Frontiers in Endocrinology, 12, 697505.

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Immortalized Human Granulosa Cell Culture

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A immortalized human primary granulosa (hGL5) cell line [51 (link)] was used for control experiments. hGL5 cells permanently overexpressing LHCGR (hGL5/LHCGR) were developed and characterized previously [9 (link),14 (link)]. Cell line were maintained in an incubator at 37 °C and 5% CO₂, in DMEM/F12 medium supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 U/mL penicillin, 50 µg/mL streptomycin (all from Gibco) and 2% ITS + Premix Universal Culture Supplement (#354352; Corning Incorporated, Corning, NY, USA).

Casarini L., Riccetti L., De Pascali F., Gilioli L., Marino M., Vecchi E., Morini D., Nicoli A., La Sala G.B, & Simoni M. (2017). Estrogen Modulates Specific Life and Death Signals Induced by LH and hCG in Human Primary Granulosa Cells In Vitro. International Journal of Molecular Sciences, 18(5), 926.

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Chondrogenic Differentiation of Expanded hPDCs

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The chondrogenic differentiation capacity of the 3D and 2D expanded hPDCs was determined based on a micromass assay as described earlier [41 (link)]. Briefly, quadruplicate 10 μl micro-masses containing 200,000 cells each were made in 24-well plates for both 2D and 3D expanded cells and incubated overnight in CM. Subsequently the medium was replaced by chondrogenic inductive medium based on DMEM-F12 (Life Technologies) supplemented with 2% FBS, 1% antibiotic–antimycotic, 1X insulin, transferrin, selenous acid (ITS+) Premix universal Culture Supplement (Corning), 100 nM Dexamethasone, 10 μM Y27632 (Axonmedchem), 50 μg/ml Ascorbic Acid, 40 μg/ml Proline (Sigma) and 10 ng/ml transforming growth factor beta 1 (TGFβ1) (Peprotech) [41 (link)]. Chondrogenic medium was refreshed every 2 days for 7 days after which the micro-masses were rinsed with PBS and fixed with ice cold methanol for 1 hour at 4°C. Subsequent rinsing steps with PBS and MiliQ water were followed by staining for 1 hour with a 0.1% Alcian Blue solution (in 0.1 M HCl). Non-specific dye was thereafter removed and a 6M guanidine hydrochloride solution was added overnight to dissolve the dye bound to the glycosaminoglycans present and quantification was performed by measuring the resulting absorbance at 620nm.

Sonnaert M., Luyten F.P., Schrooten J, & Papantoniou I. (2015). Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective. PLoS ONE, 10(8), e0136875.

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Disaggregation and Culture of Tumor Cells

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Tissue samples were disaggregated with scalpel, washed in DMEM with 1x Gibco® Antibiotic-Antimycotic solution (Thermo Fisher Scientific, USA), and fractured further by pipetting. Tumor cells were released from the tissues by enzymatic treatment with Accutase solution (Thermo Fisher Scientific, USA) for 20 min at 37°C on rotating platform in a humidified atmosphere maintained at 5% CO₂. At the end of incubation, the cells were pelleted by centrifugation (5 min, 1600 rpm). Cells were grown in DMEM-F12 (Thermo Fisher Scientific, USA) with 1% L-glutamine (Thermo Fisher Scientific, USA), 10% Fetal Bovine Serum, ES Cell-Qualified (FBS) (Thermo Fisher Scientific, USA), 1% ITS Premix Universal Culture Supplement (Corning, USA), and 0,5% Primocin™ (InvivoGen, USA) until reaching confluency. Cells above three passages on cell culture flasks were used for further experiments. All cell culture cultivations and incubations throughout the study were carried out at 37°C, 95% air, and 5% CO₂.

Megnis K., Mandrika I., Petrovska R., Stukens J., Rovite V., Balcere I., Jansone L.S., Peculis R., Pirags V, & Klovins J. (2016). Functional Characteristics of Multipotent Mesenchymal Stromal Cells from Pituitary Adenomas. Stem Cells International, 2016, 7103720.

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Tissue-Engineered Disc Replacement in Goats

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Tissue-engineered total disc replacements—eDAPS (endplate-modified Disc-Like Angle-Ply Structures)—were fabricated at a size physiologically relevant for disc replacement in goat cervical spines [Gullbrand et al., Science Translational Medicine 2018 ] by combining an AF-replacement (layers of electrospun 90 kDa PCL seeded with goat bone marrow-derived MSCs), a NP-replacement (core of 4% agarose hydrogel seeded with goat bone marrow-derived MSCs), and an acellular endplate analog (large salt-leached PCL scaffolds) according to our established methods [Gullbrand et al., Acta Biomaterialia 2018 (link); Gullbrand et al., Science Translational Medicine 2018 ]. For this study, the PCL endplate analogs were either uncoated (n = 4 eDAPS) or HA-coated (n =4 eDAPS) for 7 days. eDAPS were cultured in chondrogenic media containing 10 ng/ml TGF-β3 (Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin/streptomycin/fungizone (PSF, Antibiotic-Antimycotic; Gibco), 0.1 mM dexamethasone, 50 μg/ml Ascorbate-2-phosphate, 40 μg/ml L-Proline, 100 μg/ml sodium pyruvate, 1% ITS+ Premix universal culture supplement (Corning, 254352), 1.25 mg/ml bovine serum albumin, and 5.3 μg/ml linoleic acid-albumin) and left to grow at standard culture conditions with constant mechanical agitation until their removal from culture at 10 weeks.

Fainor M., Mahindroo S., Betz K.R., Augustin J., Smith H.E., Mauck R.L, & Gullbrand S.E. (2023). A tunable calcium phosphate coating to drive in vivo osseointegration of composite engineered tissues. Cells, tissues, organs, 212(5), 383-398.

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Chondrogenic Differentiation of Mouse Limb Bud Cells

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The mouse limb bud cell line, MLB13MYC clone 14, was used for micromass culture.^{(29 (link))} Briefly, cells were plated at a density of 2 × 10⁵ cells/7.5 μL drop and maintained in DMEM with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. After 1 day in culture, the medium was replaced with chondrogenic medium (DMEM supplemented with 1% FBS, 50 μg/mL ascorbic acid, 0.1 μM dexamethasone, 40 μg/ml L-proline, 1 mM sodium pyruvate, ITS+ Premix Universal Culture Supplement [Corning Inc., Corning, NY, USA], 100 IU/mL penicillin, and 100 μg/mL streptomycin) with or without 50 ng/mL GDF6. Culture medium was replaced every 2 to 3 days. One week after stimulation, the micromass cultures were fixed and stained with Alcian blue overnight. Quantification of Alcian blue dye was determined at 620 nm after extraction with 6M guanidine-HCl.^{(16 (link))} For each condition, 3 replicates were performed in parallel.

Wang J., Yu T., Wang Z., Ohte S., Yao R.E., Zheng Z., Geng J., Cai H., Ge Y., Li Y., Xu Y., Zhang Q., Gusella J.F., Fu Q., Pregizer S., Rosen V, & Shen Y. (2015). A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 31(4), 882-889.

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Quantification of Secreted Proteins from Senescent Preadipocytes

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Senescent preadipocytes at Day 5 and control untreated cells were cultured in conditioned medium (serum-free), supplemented with ITS Premix Universal Culture Supplement (Corning) for 24 h. The conditioned medium was collected, centrifuged to remove cell debris and stored at –80 °C for further analysis. After normalizing the concentrations of the collected medium in each condition to the number of cells/well, 100 μL of the conditioned medium was applied to Bio-Dot SF Microfiltration Apparatus (Bio-rad) using nitrocellulose membrane. The membranes were blocked using 4% BSA in tris-buffered saline with 0.1% Tween ^®® 20 Detergent (TBST) for 1 h at room temperature. The membranes were then incubated with the primary antibody prepared at 1:1000 dilution in TBST supplemented with 2% BSA for 1 h at room temperature, washed with TBST (3×) for 5 min. After washing, membranes were incubated with the secondary antibody prepared at 1:10,000 dilution in 2% BSA/TBST for 1 h, and then washed with TBST (3×) for 5 min. Signals were detected in the presence of SuperSignal^TM West Dura Extended Duration Substrate (Thermo Fisher Scientific) using ChemiDoc^TM MP imaging system (Bio-Rad). Band intensities were quantified using ImageJ software https://imagej.nih.gov.

Madani A.Y., Majeed Y., Abdesselem H.B., Agha M.V., Vakayil M., Sukhun N.K., Halabi N.M., Kumar P., Hayat S., Elrayess M.A., Rafii A., Suhre K, & Mazloum N.A. (2021). Signal Transducer and Activator of Transcription 3 (STAT3) Suppresses STAT1/Interferon Signaling Pathway and Inflammation in Senescent Preadipocytes. Antioxidants, 10(2), 334.

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