Leibovitz l 15 media

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

Leibovitz L-15 media is a cell culture medium designed to support the growth and maintenance of a variety of cell types. It is a complex mixture of amino acids, vitamins, salts, and other essential nutrients required for cell proliferation. The media is formulated to function in an environment without additional carbon dioxide supplementation.

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Leibovitz’s L-15 media Leibovitz L-15 and McCoy 5A (LM) media Leibovitz’s L-15 cell culture media Leibovitz-15 media Leibovitz L-15 L-15 media

33 protocols using leibovitz l 15 media

Encapsulation of Ovarian Cortical Tissue

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The pieces of ovarian cortical tissue (1mm³) (Figure 2A) were maintained in the final thaw solution media (Solution 4 for slow freezing, Solution 3 for vitrification) at 37°C until encapsulation (Figure 1E). The PEG core was prepared by cross-linking 8-arm PEG-VS (40 kDa, Jenkem Technology, Beijing, China) (5% w/v) with plasmin sensitive peptide (Ac-GCYK↓NSGCYK↓NSCG, MW 1525.69 g/mol, > 90% Purity, Genscript, ↓ indicates the cleavage site of the peptide). The PEG shell was prepared with 4-arm PEG-VS (20 kDa, Jenkem Technology) (5% w/v), Irgacure 2959 (Ciba, Switzerland, MW = 224.3) (0.4% w/v), and N-vinyl-2-pyrrolidone (Sigma-Aldrich, St. Louis, USA) (0.1% v/v).
The tissue was then placed in a 4µL droplet of degradable PEG core pre-cursor solution. After five minutes of crosslinking the core was transferred into 10µL of non-degradable PEG shell pre-cursor solution. The shell was cross-linked via UV light at constant intensity (4.4mW/cm², 6 minutes). Encapsulated tissue (Figures 1F, 2A) was maintained in Leibovitz L-15 media (Gibco, USA) at 37°C until implantation.

Brunette M.A., Kinnear H.M., Hashim P.H., Flanagan C.L., Day J.R., Cascalho M., Padmanabhan V, & Shikanov A. (2022). Human Ovarian Follicles Xenografted in Immunoisolating Capsules Survive Long Term Implantation in Mice. Frontiers in Endocrinology, 13, 886678.

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Maintenance of Human Cancer Cell Lines

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Human adenocarcinoma cells MDA-MB-231 (#ACC 732, DSMZ, Germany) were maintained in Leibovitz L15 media (Gibco™), supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin–streptomycin. Human adenocarcinoma cells MCF-7 (#ACC 115, DSMZ, Germany) were maintained in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific Inc., USA), supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin–streptomycin, 1-mM sodium pyruvate, and 1 × minimum essential medium nonessential amino acids in 5% carbon dioxide (CO₂). Human ovarian cancer cells CAOV-4, which is a kind gift of Prof. Zhivotovsky (Karolinska Institute, Stockholm, Sweden) (Zamaraev et al., 2018 (link)), were maintained in Dulbecco's modified Eagle medium/Ham's F-12 media (Pan-Biotech GmbH, Germany), supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin–streptomycin, and 0.0001% puromycin in 5% CO_2.

Wohlfromm F., Richter M., Otrin L., Seyrek K., Vidaković-Koch T., Kuligina E., Richter V., Koval O, & Lavrik I.N. (2021). Interplay Between Mitophagy and Apoptosis Defines a Cell Fate Upon Co-treatment of Breast Cancer Cells With a Recombinant Fragment of Human κ-Casein and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand. Frontiers in Cell and Developmental Biology, 8, 617762.

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Transfection and Imaging of ATP Sensor in HeLa Cells

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HeLa cells were plated in 2-well LabTek II chambered coverglass slides (Nunc) at a density of 100,000 cells per well. After 24 h, cells were transfected with a mitochondrially targeted ATP sensor (mitAteam 1.03 [30 (link)]) by JetPRIME (Polyplus) following the manufacturer’s protocol. Briefly, for each well, 1.6 μg of DNA was mixed with 150 μl JetPRIME buffer and 3.2 μl JetPRIME reagent and incubated for 10 min, then added drop-wise onto cells with wells containing 2 ml of media. After 4 h, cells were washed and supplemented with fresh growth medium to minimize toxicity. Cells were imaged 24 h later, in imaging media consisting of Leibovitz L-15 media (Gibco) supplemented with 10% FBS and 25 mM Glucose (Sigma). MitAteam was generated by inserting two copies of the cytochrome c oxidase subunit VIII mitochondrial targeting sequence in tandem (synthesized with the GeneArt String DNA fragment service by ThermoFisher) between the HindIII and BamHI restriction sites of the original ATeam plamids, a gift from Takeharu Nagai (AddGene, ##51958 [31 (link)]). Binding of ATP induces a conformational change that result in differences in FRET efficiencies.

Trinh A.L., Ber S., Howitt A., Valls P.O., Fries M.W., Venkitaraman A.R, & Esposito A. (2019). Fast single-cell biochemistry: theory, open source microscopy and applications. Methods and applications in fluorescence, 7(4), 044001.

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Maintenance and Transfection of Breast Cancer Cell Lines

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Hs578T cells were maintained in DMEM (Gibco, 11965084) with 10% FBS (Gibco, 10082147) and 0.01 mg/ml Insulin (Sigma-Aldrich, I0516) at 37°C in 5% CO₂. MCF7 cells were maintained in MEM (Gibco, 11095080) with 10% FBS at 37°C in 5% CO₂. HCC1569 cells were maintained in DMEM with 10% FBS at 37°C in 5% CO₂. MDA-MB-453 cells were maintained in Leibovitz L-15 media (Gibco, 11415064) supplemented with 10% FBS at 37°C without CO₂. LentiX-293T and HEK293T cells (a gift from Dr Roland Owens, NIH) were maintained in DMEM with 10% FBS at 37°C in 5% CO₂. Lipofectamine3000 (Thermofisher Scientific, L3000008) and Mirus TransIT-BrCa transfection reagent (Mirus, MIR 5504) were used for plasmid transfection in breast cancer cells. FuGENE6 (Promega, E2691) was used to transfect plasmids to LentiX-293T and HEK293T cells following the manufacturers’ protocol.

Shen B., Chapman J.H., Custance M.F., Tricola G.M., Jones C.E, & Furano A.V. (2020). Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations. eLife, 9, e51605.

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Culture of Human Breast Cancer Cell Lines

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Human adenocarcinoma cells MDA-MB-231 (purchased: #ACC 732, DSMZ, Braunschweig, Germany) were maintained in Leibovitz L15 media (Gibco^TM), supplemented with 10% heat-inactivated fetal calf serum and 1% Penicillin-Streptomycin. Human adenocarcinoma cells MCF-7 (purchased: #ACC 115, DSMZ, Braunschweig, Germany) were maintained in RPMI 1640 Phenol Red (Thermo Fisher Scientific Inc., Waltham, MA, USA), supplemented with 10% heat-inactivated fetal calf serum, 1% Penicillin-Streptomycin, 1 mM sodium pyruvate and 1 x MEM non-essential amino acids in 5% CO₂.

Richter M., Wohlfromm F., Kähne T., Bongartz H., Seyrek K., Kit Y., Chinak O., Richter V.A., Koval O.A, & Lavrik I.N. (2020). The Recombinant Fragment of Human κ-Casein Induces Cell Death by Targeting the Proteins of Mitochondrial Import in Breast Cancer Cells. Cancers, 12(6), 1427.

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Maintenance of Diverse Cell Lines

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PAC2 zebrafish fibroblasts derived from 24hpf zebrafish embryos were maintained at 28 °C in Leibovitz L-15 media (Gibco). PAC2 cells^{14 (link)} were kindly provided by Nicholas Foulkes (KIT) and Reinhard Koester (TU Braunschweig). HEK293T (Human Embryonic Kidney, CRL-1573) cells from the American Tissue Culture Collection, ATTC, Wesel, Germany were maintained at 37 °C with 5% CO₂ in DMEM media (Gibco). Primary gastric adenocarcinoma (AGS) cells were maintained at 37 °C with 5% CO₂ in RPMI-1640 media (Gibco). All media was supplemented with 10% FBS (Gibco) and 1% Pen/Strep (Gibco) and with l-Glutamine.

Brunt L., Greicius G., Rogers S., Evans B.D., Virshup D.M., Wedgwood K.C, & Scholpp S. (2021). Vangl2 promotes the formation of long cytonemes to enable distant Wnt/β-catenin signaling. Nature Communications, 12, 2058.

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Culturing Fish Cell Lines for Immunostimulation

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The RTS11 cell line was cultured in flasks (75 cm²) at 20°C in growth media (Leibovitz L-15 media + 30% FBS + 1% Penicillin/Streptomycin), the cells were generally non-adherent to the flasks prior to any stimulation. Cells were collected and pelleted by centrifugation at 500g for 10 mins at 4°C before being resuspended in fresh stimulation media (Leibovitz L-15 media + 1% FBS + 1% Penicillin/Streptomycin) and adjusted to 5x10⁵ cells/ml, then 1 ml was added to 24 well plates. RTgutGC cells were cultured in flasks at 20°C in growth media (Leibovitz L-15 media (Gibco) + 10% FBS + 1% Penicillin/Streptomycin (1% P/S) (Gibco)). Prior to stimulation, RTgutGC cells were trypsinised, once separated from the flask, cells were resuspended in growth media, washed, and plated as described for RTS11. These cells were then immunostimulated as described below in section 2.4.

Porter D., Peggs D., McGurk C, & Martin S.A. (2022). Gut Associated Lymphoid Tissue (GALT) primary cells and stable cell lines as predictive models for intestinal health in rainbow trout (Oncorhynchus mykiss). Frontiers in Immunology, 13, 1023235.

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Comparative Analysis of DENV Virion Stability

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To detect differences in virion stability between DENV-Cipro and DENV-DMEM, 7 T25 flasks of 80% confluent HEK-293 cells were infected at MOI: 0.1 with each of the DENV-Cipro or DENV-DMEM lineages (both from passage seven) or DENV-P. After 2 h of incubation with occasional rocking, the flasks were washed with 1X PBS and 5 mL of cell-specific media was added to each flask. On the expected day of peak viral titer, day 3, the viral supernatants were collected on ice then clarified by centrifugation at 1200 rpm for 10 min at 4 °C. Immediately after centrifugation, 45 μL of each clarified supernatant was added to cryotubes with 405 μL Leibovitz L15 media (Gibco, Life Technologies, Grand Island, NY) and placed into a 37 °C incubator, one tube for each virus and for each time point (0 h post-incubation, 2, 4, 6, 8, 12, 24, and 30). At each time point, the cryotube was removed from the incubator and 50 μL of 10X SPG was added before the tube was snap frozen on a dry ice-methanol bath. Frozen samples were stored at −80 °C and viral titers were determined using HEK-293 cells as described above.

Scroggs S.L., Gass J.T., Chinnasamy R., Widen S.G., Azar S.R., Rossi S.L., Arterburn J.B., Vasilakis N, & Hanley K.A. (2020). Evolution of resistance to fluoroquinolones by dengue virus serotype 4 provides insight into mechanism of action and consequences for viral fitness. Virology, 552, 94-106.

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Isolation and Dissociation of Trigeminal Ganglia

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Bilateral TGs were isolated by removing the skull cap and brain to expose the TGs readily visible in the base of the cranium. Using spring-loaded scissors the TGs were dissected and placed into cold HBSS and dissociated based on the previously described protocol^{51 (link)}. Briefly, TG were minced and incubated in HBSS containing collagenase type II (Gibco) and dispase type II (Gibco) at 37 °C for 20 min and mechanically dissociated using a fire polished glass Pasteur pipette. A 12.5% by 28% percoll gradient was used to separate myelin and nerve debris from TG neurons. Cells were then plated in DMEM/F12 (Gibco) containing 5% fetal bovine serum and antibiotics (penicillin/streptomycin, 50 U/mL) on 5 mm coverslips. Coverslips were flooded 2 h later with Leibovitz L-15 media (Gibco) containing 10% fetal bovine serum, 5 mM HEPES and 5 mM glucose, and used at room temperature. Experiments were performed within 8 h of tissue harvest.

Scheff N.N., Wall I.M., Nicholson S., Williams H., Chen E., Tu N.H., Dolan J.C., Liu C.Z., Janal M.N., Bunnett N.W, & Schmidt B.L. (2022). Oral cancer induced TRPV1 sensitization is mediated by PAR2 signaling in primary afferent neurons innervating the cancer microenvironment. Scientific Reports, 12, 4121.

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Transfection and Imaging of ATP Sensor in HeLa Cells

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