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Media 199

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Media 199 is a cell culture medium designed for the growth and maintenance of a variety of cell types. It provides a balanced formulation of nutrients, salts, and other components to support cell viability and proliferation.

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Lab products found in correlation

Cosmic calf serum, by GE Healthcare (3 mentions) Mcdb 105, by Merck Group (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Media 105, by Merck Group (2 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions) Hct116, by American Type Culture Collection (2 mentions) Sw480, by American Type Culture Collection (2 mentions) Sw620, by American Type Culture Collection (2 mentions) Hct15, by American Type Culture Collection (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) Pen strep, by Thermo Fisher Scientific (2 mentions) Media 199, by Thermo Fisher Scientific (2 mentions) Matrigel matrix, by Merck Group (2 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Epidermal growth factor (egf), by GE Healthcare (1 mentions) Primaria, by Corning (1 mentions) Rnase inhibitor, by Takara Bio (1 mentions) Hmec 1, by American Type Culture Collection (1 mentions)

16 protocols using media 199

1

Bone Marrow Aspiration and Mononuclear Cell Isolation

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Twenty two hours after stroke, the mice were anesthetized with isoflurane. An incision was made through the skin to the medial aspect of the left tibia. The periosteum was removed and the surgeon drilled a 0.5 ×0.5mm burr hole extending into the medullary cavity. A 261/2 gauge hypodermic needle was inserted into the medullary cavity and connected to a heparinized syringe. Bone marrow was aspirated while rotating and moving the needle back and forth. The medullary cavity was flushed with saline and the content aspirated. The burr hole was sealed with bone wax and the skin was closed. In the saline control group, only the needle was inserted into the medullary cavity but no content was aspirated.
The cells from the bone marrow aspirate were triturated, centrifuged, and washed in PBS + 0.5% bovine serum albumin (BSA). Cells were then suspended in Media 199 (Sigma, USA) and counted using a hemocytometer and coulter counter. The cell suspension was added on top of 5 mL Ficoll-Paque PLUS (GE, USA) in a 15mL conical vial and then centrifuged. The MNCs were collected, washed with PBS+0.5% BSA, and then counted. Cells were then suspended in sterile, iced PBS at the desired concentration before cell separation.
Yang B., Parsha K., Schaar K., Xi X., Aronowski J, & Savitz S.I. (2016). Various cell populations within the mononuclear fraction of bone marrow contribute to the beneficial effects of autologous bone marrow cell therapy in a rodent stroke model. Translational stroke research, 7(4), 322-330.
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2

Cell Line Characterization for AGCT Research

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Control cell lines were used as positive controls (MCF-7, SH-SY5Y) and to test differential response to treatment (SVOG-3e). MCF-7, an estrogen- and progesterone-receptor-positive human breast cancer cell line, was used as positive control for the anti-hormonal therapies [49 (link)]. Additionally, SH-SY5Y, a human neuroblastoma cell line, was used as positive control for chemotherapeutic agents [50 (link)]. KGN, a human AGCT cell line derived from a 63-year-old patient heterozygous for FOXL2 c.402C > G, was used as an additional AGCT cell line to evaluate potential AGCT response [51 (link)]. Finally, the immortalized human granulosa cell line SVOG-3e [52 (link)] was used to assess selective drug response in AGCT versus healthy granulosa cells. SH-SY5Y and MCF-7 were cultured in DMEM/F12 (Thermo Fischer Scientific) + 10% FBS and 1% Pen/Strep and RPMI 1640 (Thermo Fischer Scientific, Waltham, MA, USA) + 10% FBS and 1% Pen/Strep, respectively. KGN was cultured in conditions identical to AGCT-patient-derived cell lines. SVOG-3e was cultured in a 1:1 ratio of Media 199 and Media 105 (Sigma-Aldrich, St. Louis, MO, USA) with 5% FBS and 1% Pen/Strep.
Roze J., Sendino Garví E., Stelloo E., Stangl C., Sereno F., Duran K., Groeneweg J., Paijens S., Nijman H., van Meurs H., van Lonkhuijzen L., Piek J., Lok C., Jonges G., Witteveen P., Verheijen R., van Haaften G., Zweemer R, & Monroe G. (2021). In Vitro Systematic Drug Testing Reveals Carboplatin, Paclitaxel, and Alpelisib as a Potential Novel Combination Treatment for Adult Granulosa Cell Tumors. Cancers, 13(3), 368.
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3

Cultivation of Immortalized Human Colonic Cells

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Immortalized non-transformed human colonic epithelial cell lines (HCEC) were a gift from J. Shay (UT Southwestern)[51 (link)]. HCECs were grown in medium composed of 4 parts DMEM to 1 part media 199 (Sigma-Aldrich) with 2% cosmic calf serum (GE Healthcare), 25 ng/mL EGF, 1 μg/mL hydrocortisone, 10 μg/mL insulin, 2 μg/mL transferrin, 5 nM sodium selenite, and 50 μg/mL gentamycin sulfate. HCECs were grown in a hypoxia chamber with 2% O2 and 5% CO2 at 37°C.
Robb C.M., Kour S., Contreras J.I., Agarwal E., Barger C.J., Rana S., Sonawane Y., Neilsen B.K., Taylor M., Kizhake S., Thakare R.N., Chowdhury S., Wang J., Black J.D., Hollingsworth M.A., Brattain M.G, & Natarajan A. (2017). Characterization of CDK(5) inhibitor, 20-223 (aka CP668863) for colorectal cancer therapy. Oncotarget, 9(4), 5216-5232.
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4

Cultivating Colon Cancer Cell Lines

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Colon cancer cell lines HCT116 (ATCC CCL-247), LoVo (ATCC CCL-229), RKO (CRL-2577), HCT15 (ATCC CCL-225), SW480 (ATCC CCL-228), SW620 (ATCC CCL-227), and T84 (ATCC CCL-248) were obtained from American Type Culture Collection (ATCC). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). All colon cancer cells were grown with ambient O2 and 5% CO2 at 37 °C. Immortalized, non-transformed human colonic epithelial cell lines (HCEC) were kindly provided by Jerry Shay (UT Southwestern) [18 (link)]. HCEC media consists of four parts DMEM to one-part media 199 (Sigma-Aldrich) supplemented with 1 μg/mL hydrocortisone, 25 ng/mL EGF, 10 μg/mL insulin, 5 nM sodium selenite, 2 μg/mL transferrin and 2% cosmic calf serum (GE Healthcare). HCECs were grown in 2% O2 and 5% CO2 at 37 °C within an enclosed hypoxia chamber. HCECs are grown on Corning™, Primaria™ plates.
Neilsen B.K., Chakraborty B., McCall J.L., Frodyma D.E., Sleightholm R.L., Fisher K.W, & Lewis R.E. (2018). WDR5 supports colon cancer cells by promoting methylation of H3K4 and suppressing DNA damage. BMC Cancer, 18, 673.
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5

Single-cell RNA-seq of Ovarian Cancer Cell Lines

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Culture of ovarian cancer cell lines OV2295, TOV2295, and OV2295R cells were cultured in a 1:1 mix of Media 199 (Sigma Aldrich) and MCDB 105 (Sigma Aldrich) supplemented with 10% FBS in a humidified environment at 37C. For single-cell RNA sequencing, all cells used in this study were sorted on a BD Influx into 384 well plates using index-sorting and single-cell purity mode directly into lysis buffer(6,67% Polyethylene Glycol, 0.1% Triton X-100, RNAse Inhibitor (Takara), dNTPs (0.67 mM/each), and Oligo-dT (0.67uM)). Sorted plates were stored at −80 °C and thawed immediately prior to library generation. The cell line originates from ref. 27 (link).
Jun S.H., Toosi H., Mold J., Engblom C., Chen X., O’Flanagan C., Hagemann-Jensen M., Sandberg R., Aparicio S., Hartman J., Roth A, & Lagergren J. (2023). Reconstructing clonal tree for phylo-phenotypic characterization of cancer using single-cell transcriptomics. Nature Communications, 14, 982.
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6

Cell Culture Protocols for Neuroscience

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G55 cells (provided by Michael E. Sughrue, M.D. from the University of Oklahoma, Department of Neurosurgery) were cultured in DMEM (LifeTechnologies, Waltham, MA) supplemented with 10% cosmic calf serum (CCS; HyClone, Logan, UT) and 1 % penicillin/streptomycin. Noncancerous human endothelial microvascular cells (HMEC-1) were obtained from ATCC and cultured in Media-199 (SigmaAldrich) supplemented with 15% fetal bovine serum (VWR, Radnor, PA) and penicillin/streptomycin. Mouse Astrocytes, obtained from ATCC were cultured in DMEM (LifeTechnologies, Waltham, MA), and supplemented with 10% cosmic calf serum (CCS; HyClone, Logan, UT).
Ziegler J., Bastian A., Lerner M., Bailey-Downs L., Saunders D., Smith N., Sutton J., Battiste J.D., Ihnat M.A., Gangjee A, & Towner R.A. (2017). AG488 as a therapy against gliomas. Oncotarget, 8(42), 71833-71844.
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7

Porcine Cumulus-Oocyte Complex Maturation

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Ovaries were collected from the slaughterhouse immediately after slaughter and transported in a thermos. They were washed with saline solution at 38 °C upon arrival at the laboratory. A vacuum pump and an 18-gauge needle were used to aspirate cumulus-oocyte complexes (COCs). A follicle measuring more than 3 mm was chosen as the suction target. The aspirate was placed in a 50 mL tube and allowed to settle for 15 min before washing with T-HEPES (media 199, Sigma-Aldrich; 1% PVA, Sigma-Aldrich; sodium pyruvate, Sigma-Aldrich; 200 µM D-glucose; 3mM penicillin/streptomycin, Gibco 1835954; 1% sodium bicarbonate, s8875, Sigma-Aldrich). The sediment and T-HEPES mixture were poured into a 60 mm petri dish to locate COCs using a microscope. The collected COCs were washed three times in in vitro maturation media (media 199, Gibco; 10% porcine follicular fluid, EGF 10 ng/mL, HCG 10 IU/mL, cAMP 100 μM, and PMSG 10 IU/mL) and cultured in a four-well dish. COCs were allowed to mature for 42 h, which consisted of culturing them for 22 h in hormone-supplemented media and for 20 h in hormone-free media.
Yoon S., Lee S., Park C., Choi H., Yoo M., Lee S.C., Hyun C.H., Kim N., Kang T., Son E., Ghosh M., Son Y.O, & Hur C.G. (2022). An Efficacious Transgenic Strategy for Triple Knockout of Xeno-Reactive Antigen Genes GGTA1, CMAH, and B4GALNT2 from Jeju Native Pigs. Vaccines, 10(9), 1503.
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8

Isolation and Culture of Neonatal Rat Ventricular Myocytes

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NRVM were isolated according to our established protocols using a standard isolation system (Worthington Biochemical Corporation). In brief, hearts from 1–3 day old Sprague Dawley rats were harvested, minced and digested with trypsin for 16–20 hours. The next day, trypsin inhibitor was added and the tissue was further digested with collagenase. Cells were released by trituration, filtered twice, incubated for 20 min and pelleted by centrifugation. After resuspension cells were seeded in plating medium [Dulbecco’s Modified Eagle Medium (DMEM; Cellgro) with 17% Media 199 (Invitrogen), 15% fetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin solution (P/S; Invitrogen)] on laminin-coated glass cover slips in 24-well plates. After 72 hours, cells were further cultured in maintenance media [DMEM with 20% Media 199, 1% insulin-transferrin-sodium selenite solution (ITS; Sigma-Aldrich) and 1% P/S] in the presence of 100 μM 5-bromo-2′ deoxyuridine (BrdU; Sigma-Aldrich) for another 4 days and then treated with recombinant murine FGF23 (6His-tagged Tyr25-Val251 [Arg179Gln]; 26.1 kDa) or FGF2 (Ala11-Ser154; 16.2 kDa) (both R&D Systems) at 25 ng/ml for different time points. For treatment studies, an FGFR4 blocking antibody (human monoclonal, U3-11; U3Pharma) was used at 10 μg/ml.
Grabner A., Schramm K., Silswal N., Hendrix M., Yanucil C., Czaya B., Singh S., Wolf M., Hermann S., Stypmann J., Di Marco G.S., Brand M., Wacker M.J, & Faul C. (2017). FGF23/FGFR4-mediated left ventricular hypertrophy is reversible. Scientific Reports, 7, 1993.
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9

Immortalized Ovarian Surface Epithelial Cells

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Transfection of human ovarian surface epithelial cells with the SV40 early region expressing T/t antigen and then subsequent infection with a retrovirus containing a full-length hTERT cDNA, to create the IOSE-29 (Immortalized Ovarian Surface Epithelial-29) cell line has been previously described [27 (link),28 (link)]. For this study, IOSE-29 cells were cultured in 10 cm dishes with 10 ml of ovarian epithelial-cell culture medium consisting of 1:1 MCDB105 and Media 199 (Sigma-Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Life Technologies, Inc.).
Patel P.R., Hegde M.L., Theruvathu J., Mitra S.A., Boldogh I, & Sowers L. (2015). Norepinephrine Reduces Reactive Oxygen Species (ROS) and DNA Damage in Ovarian Surface Epithelial Cells. Journal of bioanalysis & biomedicine, 7(3), 75-80.
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10

Culturing Cell Lines for Research

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The 184-hTERT cell lines were cultured at 37 °C, 5% CO2, in serum-free mammary epithelial cell basal media (MEBM, Lonza), supplemented with mammary epithelial cell growth media single quots (Lonza), 5 μg/ml transferrin (Sigma), 1.25 M of isoproterenol (Sigma Aldrich). HCT116 cell lines were cultured at 37 °C, 5% CO2, in McCoys 5A media (Sigma Aldrich) supplemented with 10% FBS (Sigma Aldrich). The TOV3133D and TOV3133G cell lines were cultured at 37 °C, 5% CO2, in a 1:1 mix of media 199 (Sigma Aldrich) and media 105 (Sigma Aldrich) supplemented with 10% FBS.
Farahani H., de Souza C.P., Billings R., Yap D., Shumansky K., Wan A., Lai D., Mes-Masson A.M., Aparicio S, & P. Shah S. (2017). Engineered in-vitro cell line mixtures and robust evaluation of computational methods for clonal decomposition and longitudinal dynamics in cancer. Scientific Reports, 7, 13467.
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