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M199 media

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M199 media is a cell culture medium primarily used for the maintenance and growth of a variety of mammalian cell types. It provides the necessary nutrients and growth factors required for cell survival and proliferation in vitro.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Heparin, by Merck Group (2 mentions) Aria 2 flow cytometer, by BD (2 mentions) Collagenase 2, by Merck Group (2 mentions) Dnase, by Worthington (2 mentions) Endothelial growth media, by Lonza (2 mentions) Streptomycin, by Merck Group (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Bme vitamins, by Merck Group (2 mentions) Penicillin streptomycin, by Merck Group (2 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) Hepes, by Merck Group (2 mentions) Hepes ph 7, by Merck Group (2 mentions) Glutamine, by Merck Group (1 mentions) α mem, by Merck Group (1 mentions) Gelatin, by Merck Group (1 mentions) Huvecs, by ScienCell (1 mentions)

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M199 culture media

34 protocols using m199 media

1

Culturing UCX and HUVEC Cells

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UCX® were cultured in static monolayers in α-MEM with 1 g/L glucose and 2 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA), hereafter designated basal medium (BM), supplemented with 20 % foetal bovine serum (FBS; Gibco®, Madrid, Spain), in a humidified incubator at 37 °C and 7 % CO2.
Human umbilical vein endothelial cells (HUVECs) (Sciencell, Carlsbad, CA, USA) were cultured in M199 media (Sigma-Aldrich, St. Louis, MO, USA), hereafter designated endothelial basal medium (EBM) supplemented with 10 % FBS (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA), 50 μg/ml endothelial cell growth supplement (ECGS) (Sigma-Aldrich), 100 μg/ml heparin (Sigma-Aldrich) and 1 % penicillin-streptomycin (10,000 U/mL, Sigma-Aldrich), hereafter designated endothelial growth medium (EGM). Cells were grown in flasks coated with 0.2 % gelatin (Sigma-Aldrich) until 70 % confluence and used up to passage 6.
Pereira A.R., Mendes T.F., Ministro A., Teixeira M., Filipe M., Santos J.M., Bárcia R.N., Goyri-O’Neill J., Pinto F., Cruz P.E., Cruz H.J, & Santos S.C. (2016). Therapeutic angiogenesis induced by human umbilical cord tissue-derived mesenchymal stromal cells in a murine model of hindlimb ischemia. Stem Cell Research & Therapy, 7, 145.
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2

Feeding Bartonella quintana to Lice

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Bartonella quintana were harvested from a single chocolate agar plate using a sterile loop and suspended into 1 ml PBS (0.1 M, pH 7.2, 1 plate/ml PBS). Spectrophotometric readings (OD600) were taken for B. quintana suspended in PBS to approximate cell counts per ml of blood. B. quintana cells were pelleted by centrifugation at 1000 g for 4 min, and resuspended in 100 μl of fresh PBS to remove residual media. A 10-μl aliquot of the suspension was serially diluted into M199 media (Sigma Chemicals), supplemented with glutamine, sodium pyruvate and 20% fetal bovine serum (M199S), and dilutions (10−4–10−8 ml PBS suspension/ml M199S) were plated in triplicate for CFU/ml blood determination. The remaining suspension was then mixed with 4 ml of human blood without antibiotics to obtain a titre of ~1 × 107 CFU/ml (Kosoy et al., 2004 (link)) and used to fill the blood reservoir of each feeding unit. Approximately 100 lice, which had been starved for 6 h, were fed on B. quintana-infected blood using the in vitro rearing system for 18 h (overnight). Following feeding, lice were transferred to a new rearing unit with non-infected blood for the remainder of the experiments. Mortality data were also recorded for all experiments. Five replicated experiments for both head and body lice were carried out.
Previte D., Olds B.P., Yoon K., Sun W., Muir W., Paige K.N., Lee S.H., Clark J., Koehler J.E, & Pittendrigh B.R. (2014). Differential gene expression in laboratory strains of human head and body lice when challenged with Bartonella quintana, a pathogenic bacterium. Insect molecular biology, 23(2), 244-254.
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3

Isolation and Culture of Ca2+-Tolerant Rat Cardiomyocytes

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The isolation and culture of Ca2+-tolerant myocytes from each rat heart followed the protocol described earlier (Lang et al., 2015 (link), 2017 (link)). Briefly, Ca2+-tolerant adult Sprague–Dawley rat or F344BN rat myocytes were isolated from collagenase-treated hearts (Lang et al., 2015 (link), 2017 (link)). Rod-shaped myocytes were plated on laminin-coated coverslips in Dulbecco’s modified Eagle’s medium supplemented with 5% FBS plus P/S at 37°C for 2 h (Lang et al., 2015 (link), 2017 (link)) and then replaced by serum-free Dulbecco’s modified Eagle’s medium plus P/S media containing recombinant AdGFP, AdPKCα, or AdPKCαDN or media alone (NT). After an additional hour, M199 media (Sigma-Aldrich) supplemented with 10 mM HEPES, 0.2 mg/ml BSA, and 10 mM glutathione plus P/S (M199+) was added to each coverslip. For contractile function studies, rat myocytes were paced starting the day after isolation, with media exchanged every 12 h. Media were also changed daily for myocytes used in protein expression work.
Ravichandran V.S., Patel H.J., Pagani F.D, & Westfall M.V. (2019). Cardiac contractile dysfunction and protein kinase C–mediated myofilament phosphorylation in disease and aging. The Journal of General Physiology, 151(9), 1070-1080.
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4

Regulation of Mesothelial Cell Signaling

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Human mesothelial cells (MET5A) (Cat No. CRL‐9444, ATCC) were cultured in M199 media (Sigma‐Aldrich Canada Ltd). In the first experiment, human MET5A cells were transfected with siRNA against Ror2 (Stealth, Invitrogen) or a non‐targetting siRNA (Fisher Scientific) for 16 hours. Following this, serum‐starved cells were treated with AdWNT5A (10 MOI) for 24 hours. In the following experiment, human MET5A cells were transfected with small interfering RNA (siRNA) against Ror2 (Stealth, Invitrogen) or non‐targetting siRNA (Fisher Scientific). Twenty‐four hours after transfection, serum‐starved cells were treated with rTGFB (R&D Systems) at 2.5 ng/mL or PBS for 8 hours. Cells were harvested to collect supernatant, protein and RNA.
Padwal M., Liu L, & Margetts P.J. (2020). The role of WNT5A and Ror2 in peritoneal membrane injury. Journal of Cellular and Molecular Medicine, 24(6), 3481-3491.
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5

Isolation and Culture of Primary Islets

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Primary islets were isolated using an intraductal collagenase technique [28 (link)]. Briefly, after clamping the common bile duct at its entrance to the duodenum, pancreas was injected with 3 ml of a collagenase P (1mg/ml, Roche) solution in M199 media (Sigma). Dissected pancreas was then digested for 17 min at 37°C, after which they were disrupted by shaking for 30 seconds. Islets were subsequently purified through 100 μm wire mesh and Histopaque 1077 (Sigma) density centrifugation. Islets were cultured for 1-2 hours at 37°C in RPMI 1640 media (Sigma) supplemented with 10% FBS (26140, Life Technologies), 1% Penicillin and Streptomycin (Life Technologies), handpicked under inverted bright-field microscopy, and allowed to recover overnight at 37°C and 5% CO2. Islets were serum and glucose starved (2.8 mM glucose RPMI 1640 without FBS) for 2 hours and then stimulated with 2.8 mM and 25 mM glucose for 1 hour.
Sato K., Idelevich A., Nagano K., Rowe G.C., Gori F, & Baron R. (2017). Hypothalamic ΔFosB prevents age-related metabolic decline and functions via SNS. Aging (Albany NY), 9(2), 353-369.
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6

Isolation of Mouse Cardiomyocytes

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Isolation of mouse cardiomyocytes was modified from a previously published procedure [31 (link)]. Hearts were excised from animals anesthetized with sodium pentobarbital and their ascending aorta was cannulated and mounted on a perfusion aperture to allow perfusion of coronary vasculature. Hearts were perfused for 4 min with cell isolation buffer that contained (in mM): 113 NaCl, 4.7 KCl, 1.2 MgSO4, 0.6 NaH2PO4, 0.6 KH2PO4 10 2,3- butanedione monoxime, 30 taurine, and 20 glucose. Subsequently, cell isolation buffer containing 620 U/ml collagenase, 0.015 mg/ml DNase I, and 0.104 mg/ml protease XIV was perfused for 10 minutes. Digested hearts were triturated in the collagenase-containing buffer to free individual myocytes. Cardiomyocytes were plated on laminin-coated glass slides (Millicell EZ slide, Millipore, Millipore Billerica, MA) in M199 media (Sigma, St. Louis, MO) containing 0.02% BSA and 5% fetal bovine serum.
Strakova J., Dean J.D., Sharpe K., Meyers T.A., Odom G, & Townsend D. (2014). Dystrobrevin increases dystrophin's binding to the dystrophin-glycoprotein complex and provides protection during cardiac stress. Journal of molecular and cellular cardiology, 76, 106-115.
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7

Isolation and Characterization of Human Adipose-Derived Vessel-Forming Progenitors

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Human adipose lipoaspirates were obtained after informed consent from female patients (26–54 years old). Fresh lipoaspirates were separated from blood, and lipoaspiration fluid were passed through a 100 μm nylon mesh. Solid matter was then resuspended in collagenase digest buffer (2.2 mg/mL Collagenase II [Sigma] supplemented with 100 U/mL DNase [Worthington] in M199 Media [Sigma]), shaken in CERTOMAT BS-1 Incubation-Shaking Cabinet at 37 °C for 30 minutes, sedimented on Histopaque-1119 Density gradient to remove blood and dead cells, washed, and pelleted at 400×g at 4 °C. Cells were then plated on Lonza Endothelial Growth Media and transduced with CMV-GFP expressing lentivirus. Twenty-four hours after transduction, cells were lifted with collagenase, resuspended in staining media (2% FBS in PBS), blocked with rat IgG, and stained with fluorochrome-conjugated antibodies against human CD45, Tie2, PDGFRα, and CD31 for FACS on the BD Aria II Flow Cytometer. As with mouse samples, the gating was adjusted based on compensation, fluorescence minus one controls and, unstained cells. In ischemic rescue studies, 500 000 sorted VSPC1+VSPC2 vessel-forming progenitors were injected around ligated femoral artery of 8- to 12-week-old immunodeficient Rag2/gamma(c) knockout male mice.
Zhao L., Lee A.S., Sasagawa K., Sokol J., Wang Y., Ransom R.C., Zhao X., Ma C., Steininger H.M., Koepke L.S., Borrelli M.R., Brewer R.E., Lee L.L., Huang X., Ambrosi T.H., Sinha R., Hoover M.Y., Seita J., Weissman I.L., Wu J.C., Wan D.C., Xiao J., Longaker M.T., Nguyen P.K, & Chan C.K. (2023). A Combination of Distinct Vascular Stem/Progenitor Cells for Neovascularization and Ischemic Rescue. Arteriosclerosis, Thrombosis, and Vascular Biology, 43(7), 1262-1277.
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8

Cultivation of Leishmania Parasites

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L. infantum isolate M/CAN/ES/98/10445 (zymodeme MON-1) was kindly provided by Dr. Alfredo Toraño (Instituto de Salud Carlos III Majadahonda, Spain) and cultured at 27°C in RPMI 1640 supplemented with l-glutamine (Gibco, Waltham, MA, USA), 10% heat inactivated fetal calf serum (Gibco, Waltham, MA, USA), as well as 100 μg/ml streptomycin and 100 IU/ml penicillin (Cambrex, East Rutherford, NJ, USA). L. major promastigotes (LV39 line Rho/SU/59/P clone 5) were grown at 27°C in M199 media (Sigma–Aldrich, St Louis, MO, USA) supplemented with 20% heat-inactivated fetal calf serum. Transfected parasites were grown in the presence of 100 μg/ml G418 (Gibco, Waltham, MA, USA).
Fernández-Orgiler A., Martínez-Jiménez M.I., Alonso A., Alcolea P.J., Requena J.M., Thomas M.C., Blanco L, & Larraga V. (2016). A putative Leishmania DNA polymerase theta protects the parasite against oxidative damage. Nucleic Acids Research, 44(10), 4855-4870.
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9

AKAP150 Localization in Cardiac Myocytes

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Isolated cardiac myocytes from WT and AKAP150−/− mice were plated on BD Cell-Tak coated cover slips. Cells were allowed to attach for 4 hrs. at 37 °C in M199 media (Sigma-Aldrich). Following removal of media cells were rinse twice with PBS and fixed in 1% paraformaldehyde, washed three times in PBS, and permeabilized with 0.075% Triton X-100/PBS solution. Following permeabilization cells were incubated in blocking buffer containing 2% donkey serum, 20% goat serum, and 1% bovine serum albumin in antibody dilution buffer (0.1% Triton X-100 and 1% IgG free BSA in PBS). Cells were incubated overnight (4 °C) with primary antibodies; mouse anti-α-actinin (A7811, Sigma-Aldrich, 1:500) and goat anti-AKAP150 (AKAP150-C20, sc-6445, Santa Cruz Biotech, 1:500). Secondary antibodies Alexa Fluor 488-congugated donkey anti-mouse and Alexa Fluor 568-conjugated donkey antigoat (Molecular Probes) were use to detect α-actinin and AKAP150 respectively (1h at 27°C). Secondary Ab incubation was followed by PBS wash and the cells were mounted on slides. Primary antibodies were omitted in negative control experiments to test labeling specificity. Cells were visualized (512×512 pixel images) using a Radiance 2100 confocal system coupled to a Nikon TE300 inverted microscope equipped with a Nikon 60x oil immersion lens (NA=1.4) lens and a zoom of 3.5 (pixel size=0.1 µm).
Nieves-Cintrón M., Hirenallur-S D., Nygren P.J., Hinke S.A., Dell’Acqua M.L., Langeberg L.K., Navedo M., Santana L.F, & Scott J.D. (2015). AKAP150 participates in calcineurin/NFAT activation during the down-regulation of voltage-gated K+ currents in ventricular myocytes following myocardial infarction. Cellular signalling, 28(7), 733-740.
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10

In vitro cultivation of Babesia ovata

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Bovine blood was purchased from Japan Bio Serum (Tokyo, Japan) for the in vitro cultivation of the intraerythrocytic parasites, Babesia ovata (Miyake strain; Minami and Ishihara, 1980 ). After preparing red blood cells (RBCs), B. ovata was cultivated with RBCs in a sterilized mixture of GIT (Fujifilm Wako Pure Chemical, Osaka, Japan) and M199 media (Sigma-Aldrich, MO, USA) under a low-oxygen atmosphere, 5% O2, 5% CO2, and 90% N2, at 37°C as described previously (Igarashi et al., 1994 (link); Maeda et al., 2016 (link)).
Kuniyori M., Sato N., Yokoyama N., Kawazu S.I., Xuan X., Suzuki H., Fujisaki K, & Umemiya-Shirafuji R. (2022). Vitellogenin-2 Accumulation in the Fat Body and Hemolymph of Babesia-Infected Haemaphysalis longicornis Ticks. Frontiers in Cellular and Infection Microbiology, 12, 908142.
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