Nu serum 1

Manufactured by BD

Sourced in United Kingdom, United States

Nu-Serum I is a laboratory reagent designed for use in various research and analytical applications. It serves as a general-purpose serum supplement to support the growth and maintenance of cell cultures. The product provides a balanced mixture of nutrients, growth factors, and other essential components required for cell proliferation and differentiation.

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Lab products found in correlation

14 protocols using nu serum 1

Metformin Regulation of Adrenocortical Cell Metabolism

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Human adrenocortical NCI-H295R cells were obtained from American Type Culture Collection (ATCC; CRL-2128). H295R cells were cultured in DMEM/Ham’s F-12 medium containing L-glutamine and 15 mM HEPES medium (GIBCO, Paisley, UK) supplemented with 5% NU-I serum (BD Biosciences, Allschwil, Switzerland), 0.1% insulin, transferrin, and selenium (100 U/ml; GIBCO), penicillin (100 U/ml; GIBCO) and streptomycin (100 µg/ml; GIBCO). The serum-free starvation medium consisted of DMEM/Ham’s F-12 medium, penicillin and streptomycin (100 µg/ml; GIBCO). For microarray experiments and for steroid profiling experiments, cells were first grown in normal growth medium for 24 h. Medium was then replaced, and cells were grown in the presence of 10 mM metformin in serum-free medium for 48 h^{21 (link)}.

Udhane S.S., Legeza B., Marti N., Hertig D., Diserens G., Nuoffer J.M., Vermathen P, & Flück C.E. (2017). Combined transcriptome and metabolome analyses of metformin effects reveal novel links between metabolic networks in steroidogenic systems. Scientific Reports, 7, 8652.

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Cell Culture Protocol for Adrenocortical and Leydig Cells

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Human adrenocortical NCI-H295R cells were purchased from American Type Culture Collection (ATCC; CRL-2128). H295R cells were cultured in DMEM/Ham’s F-12 medium containing L-glutamine and 15 mM HEPES medium (GIBCO, Paisley, UK) supplemented with 5% NU-I serum (BD biosciences), 0.1% insulin, transferrin, and selenium (100 U/ml; GIBCO), penicillin (100 U/ml; GIBCO) and streptomycin (100 μg/ml; GIBCO). The serum free starvation medium consisted of DMEM/Ham’s F-12 medium, penicillin and streptomycin (100 μg/ml; GIBCO). Mouse Leydig (MA-10) cells were kindly provided by Prof. Brigitte M. Frey, Bern, Switzerland. MA-10 cells were cultured in Waymouth medium (Sigma–Aldrich) and supplemented with 15% horse serum (GIBCO), penicillin (100 U/ml; GIBCO) and streptomycin (100 μg/ml; GIBCO). For MA-10 cells, culture dishes were pre-coated with 0.1% gelatin (Sigma–Aldrich). The serum free starvation medium of MA-10 consisted of Waymouth medium and antibiotics. Charcoal treatment of NU-I serum was performed by adding charcoal-dextran coated powder (1 g) to NU-I serum (50 mL) while gently mixing on a shaker table overnight. The followingday, charcoal-stripped serum was obtained by centrifugation at 2’000 g for 15 minutes and filtration through a 0.2 μm filter.

Udhane S.S., Pandey A.V., Hofer G., Mullis P.E, & Flück C.E. (2015). Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis. Scientific Reports, 5, 10132.

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Cell Culture Protocols for Placental, Adrenocortical, and Kidney Cells

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Human placental JEG3 cells [86 (link)] were purchased from American Type Culture Collection (ATCC) (ATCC: HTB-36™) and cultured in minimal essential medium (MEM) with Earle’s salts (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mM GIBCO), 1% penicillin (100 U/ml; GIBCO), and streptomycin (100 µg/mL; (Thermo Fisher Scientific, Waltham, MA, USA)). Human adrenocortical NCI-H295R (NCI-H295R) cells [53 (link),87 (link),88 (link)] were purchased from ATCC (ATCC: CRL-2128) and grown in DMEM/Ham’s F-12 medium containing L-glutamine and 15 mM HEPES ((Thermo Fisher Scientific, Waltham, MA, USA)) supplemented with 5% NU-I serum (Becton Dickinson, Franklin Lakes, NJ USA), 0.1% insulin, transferrin, selenium (100 U/mL; (Thermo Fisher Scientific, Waltham, MA, USA)), 1% penicillin (100 U/mL; (Thermo Fisher Scientific, Waltham, MA, USA), and streptomycin (100 μg/mL; GIBCO) and passage numbers during the experiments remained below 30 according to established protocols [40 (link),41 (link),55 (link),56 (link),89 (link),90 (link)]. Human embryonic kidney HEK293 cells (ATCC: CRL-1573) [91 (link)] were grown in DMEM GlutaMAX ^TM medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotic mix (100 ×), and 1 mM sodium pyruvate.

Castaño P.R., Parween S, & Pandey A.V. (2019). Bioactivity of Curcumin on the Cytochrome P450 Enzymes of the Steroidogenic Pathway. International Journal of Molecular Sciences, 20(18), 4606.

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NCI-H295R Cell Culture and Treatments

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Human adrenal NCI-H295R cells purchased from American Type Culture Collection (ATCC; CRL-2128) were maintained under normal growth conditions (growth medium, GM) in DMEM/Ham’s F-12 medium containing L-glutamine and 15mM HEPES medium (GIBCO, Paisley, UK) supplemented with 5% NU-I serum (Becton Dickinson, Franklin Lakes, NJ USA), 0,1% insulin, transferrin, and selenium (100U/ml; GIBCO), 1% penicillin (100 U/ml; GIBCO) and streptomycin (100μg/ml; GIBCO). The serum-free NCI-H295R medium (starvation medium, SM) contained DMEM/Ham’s F-12 medium, penicillin (100 U/ml; GIBCO), and streptomycin (100 μg/ml; GIBCO) only. The cultures were kept at 37°C with 5% CO_2, and the cells were divided once a week. For steroid profiling, mRNA expression and protein expression experiments, cells with passage numbers 18 to 23 were subcultured for 24 hours in GM on 6-well plates and then starved, or alternatively treated with metformin and resveratrol. Metformin was dissolved in water and used at a final concentration of 1mM. Resveratrol was dissolved in DMSO and used at final concentrations of 5μM to 50μM.

Marti N., Bouchoucha N., Sauter K.S, & Flück C.E. (2017). Resveratrol inhibits androgen production of human adrenocortical H295R cells by lowering CYP17 and CYP21 expression and activities. PLoS ONE, 12(3), e0174224.

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Assessing SIRT-Mediated Steroidogenesis

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H295R cells were transfected with cDNA expressing vectors for hSIRT1, hSIRT3, hSIRT5 or with the empty vector pcDNA3. Transient transfection was carried out in 12-well plates for 6 h (Falcon 3047; Becton Dickinson) using Lipofectamin 2000 reagent (Invitrogen) according to the manufacturer’s recommendations. The transfection was performed in DMEM/Ham’s F-12 medium containing L-glutamine and 15mM HEPES medium (GIBCO, Paisley, UK) supplemented with 5% NU-I serum (Becton Dickinson, Franklin Lakes, NJ USA) and 0,1% insulin, transferrin, and selenium (100U/ml; GIBCO). After 6 hours of transfection, the transfection medium was replaced by GM for 24 hours. In the following 24 hours the cells were either grown under GM or SM conditions, or under RSV treatment. Before closing the experiment 48 hours after transfection, steroidogenesis was labeled by adding [³H]-pregnenolone for 90min. Supernatants were then collected to assess the steroid profile by TLC, and cells were washed with phosphate buffered saline (PBS) for Western blot analysis.

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Culturing Human Placental and Adrenal Cells

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Cells were cultured according to established protocols^{59 (link),60}. Human placental JEG3 cells were cultured in minimal essential medium (MEM) with Earle’s salts (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mM GIBCO), 1% penicillin (100 U/ml; GIBCO), and streptomycin (100 μg/mL; Thermo Fisher Scientific). Human adrenocortical NCI-H295R (NCI-H295R) cells were grown in DMEM/Ham’s F-12 medium containing L-glutamine and 15 mM HEPES (Thermo Fisher Scientific) supplemented with 5% NU-I serum (Becton Dickinson), 0.1% insulin, transferrin, selenium (100 U/mL; Thermo Fisher Scientific), 1% penicillin (100 U/mL; Thermo Fisher Scientific), and streptomycin (100 μg/mL; GIBCO) and passage numbers during the experiments remained below 30.

Jensen S.B., Thodberg S., Parween S., Moses M.E., Hansen C.C., Thomsen J., Sletfjerding M.B., Knudsen C., Del Giudice R., Lund P.M., Castaño P.R., Bustamante Y.G., Velazquez M.N., Jørgensen F.S., Pandey A.V., Laursen T., Møller B.L, & Hatzakis N.S. (2021). Biased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase. Nature Communications, 12, 2260.

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Nicotinamide Inhibits Adrenal Cell Function

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Human adrenal NCI‐H295R cells were maintained under normal growth conditions (growth medium, GM) in DMEM:F‐12 medium (HyClone Corporation) with 0.1% Insulin‐Transferrin‐Selenium Supplement (Gibco), 2.5% Nu‐Serum I (Becton Dickinson) and 100 U/mL penicillin and streptomycin (HyClone Corporation). The serum‐free medium (starvation medium, SM) only contained DMEM:F‐12 and 100 U/mL penicillin and streptomycin. Cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C and the cells were divided once a week. Nicotinamide (Beyotime Biotechnology) was dissolved in distilled water and used at a final concentration of 1–25 mmol/L. Cells were subcultured in 12‐well plates at a density of 5 × 10⁵ cells/well, in a volume of 1.0 mL medium. When the cells were at 60%–70% confluence, the medium was replaced with starvation medium supplemented with 1, 5, or 25 mmol/L (n = 3) of NAM. The untreated control group was treated with the starvation medium supplemented with the same volume of distilled water. After 24 hours, the cell culture medium was removed and stored at −80°C until hormone analysis. The remaining cells were lysed in RIPA buffer for subsequent protein quantification. Cells treated with 25 mmol/L as the highest concentration were used for RNA isolation and RNA‐sequencing.

Gao X., Yu Z., Yang J., Gao Y., Li S, & Zhang W. (2020). An integrated RNA‐Seq and network study reveals the effect of nicotinamide on adrenal androgen synthesis. Clinical and Experimental Pharmacology & Physiology, 47(5), 821-830.

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Baicalin Modulates Steroidogenesis in NCI-H295R Cells

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The NCI-H295R human adrenocortical cell line (CRL-2128; ATCC) is considered to be a well-established model for studying steroidogenesis (Kempná et al. 2015) (link) and chosen for the in vitro study in our research. The cells were cultured under standard conditions in Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 2.5% Nu-serum I and 1% ITS premix (Becton Dickinson and Company). Baicalin (purity ≥95%), purchased from Tokyo Chemical Industry, was dissolved in 0.1% (v/v) DMSO and diluted in a range from 0.1 to 400 μmol/L. The halfmaximal inhibitory concentration (IC 50 ) was determined as described previously (Ghidini et al. 2015) (link). Besides, the cell supernatant was carefully collected from each well, without disturbing the cells, and the testosterone level was determined using an ELISA kit (R&D Systems) following the manufacturer's instructions. Finally, the most suitable concentration and intervention time for Baicalin treatment of NCI-H295R cells were determined.

Yu J., Liu Y., Zhang D., Zhai D., Song L., Cai Z, & Yu C. (2019). Baicalin inhibits recruitment of GATA1 to the HSD3B2 promoter and reverses hyperandrogenism of PCOS. The Journal of endocrinology.

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Aqueous Phase for NCI-H295A Cells

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The aqueous phase was prepared by mixing cell culture media and red food coloring in a 20:1 volumetric ratio. The cell culture media (designed for NCI-H295A cells) consisted of phenol red-free DMEM/F-12 medium (Sigma–Aldrich) containing 5% (vol/vol) Nu-Serum I (BD Biosciences), 1% ITS+ Premix (BD Biosciences), 100 U/mL penicillin, and 100 μL/mL streptomycin. Red food coloring contained water, glycerine, RD&C Red #40, citric acid, and sodium benzoate.

Lee U.N., Berthier J., Yu J., Berthier E, & Theberge A.B. (2019). Stable biphasic interfaces for open microfluidic platforms. Biomedical microdevices, 21(1), 16.

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Radiation-Induced cMET Pathway Activation in H295R ACC Cells

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The NCI-H295R human ACC cell line was obtained from the American Type Culture Collection (Manassas, VA; catalog no. CRL-2128). NCI-H295R cells were grown in Dulbecco’s modified Eagle’s medium–Ham’s F12 medium supplemented with 5% Nu-Serum I (BD Biosciences, San Jose, CA), ITS (BD Biosciences; 0.00625 mg/ml insulin, 0.00625 mg/ml transferrin, 6.25 ng/ml selenium, 1.25 mg/ml bovine serum albumin and 0.00535 mg/ml linoleic acid) and antibiotic-antimycotic solution (Corning Cellgro; 100 IU/ml penicillin, 100 µg/ml streptomycin and 250 ng/ml amphotericin B). Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously (19 ). The total number of live cells was determined by multiplying the number of cells counted with a Coulter counter by the percentage of live cells in the cell population as determined by Trypan blue dye exclusion. For radiation experiments H295R cells were irradiated at room temperature with a Mark I 137Cs irradiator (JL Shepherd & Associates, San Fernando, CA) at a dose rate of 3.5 Gy/minute (8Gy). Protein lysates were collected at different time points after irradiation (1, 3, 6, 12 and 30 hours). Protein level of cMET and phospho-cMET was evaluated by Western blot analysis.

Phan L.M., Fuentes-Mattei E., Wu W., Velazquez-Torres G., Sircar K., Wood C.G., Hai T., Jimenez C., Cote G.J., Ozsari L., Hofmann M.C., Zheng S., Verhaak R., Pagliaro L., Cortez M.A., Lee M.H., Yeung S.C, & Habra M.A. (2015). Hepatocyte growth factor/cMET pathway activation enhances cancer hallmarks in adrenocortical carcinoma. Cancer research, 75(19), 4131-4142.

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