Leibovitz l 15 medium

Manufactured by Merck Group

Sourced in United States, Germany, Belgium, Poland

Leibovitz L-15 medium is a cell culture medium formulated for use in cell and tissue culture applications. It is a basal medium that provides essential nutrients and amino acids required for cell growth and maintenance. The medium is designed to support the culture of various cell types, including adherent and suspension cells, in a controlled laboratory environment.

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Spelling variants of this product

L-15 medium (96 citations) Leibovitz’s L-15 medium (51 citations) Leibovitz L-15 (10 citations) Leibovitz-15 medium (8 citations) Leibovitz medium (8 citations) Leibovitz's L-15 medium (L-15) (4 citations) Leibovitz-15 medium (L-15 medium) (2 citations) Leibovitz-15 (L-15) growth medium (2 citations)

38 protocols using leibovitz l 15 medium

Detecting DENV in Urine and Saliva

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C6/36 cells were used in attempt to isolate DENV from urine and saliva samples. Before inoculation, urine samples were dialyzed and concentrated in PBS using the 100K Microsep ultrafiltration system (Pall, USA) and then filtered through 0.2-μm membrane (Nalgene Thermo Scientific, USA). Saliva diluted in VTM as well as urine samples were diluted 1/2 with L15 Leibovitz Medium (Sigma Aldrich, Germany) containing 2% of fetal calf serum (Gibco Life Technology, USA). Final volumes of 300 µl of diluted specimens, or controls, were inoculated into 12-well plates containing 100% confluent C6/36 cells and incubated for 1 hour at 28°C. After incubation, 1.7 ml of medium was added to each well and the plates were incubated at 28°C. After 7 days, cells were harvested and DENV was detected by an immunofluorescence assay using serotype-specific monoclonal antibodies as described previously [33 (link)]. For samples that tested negative, two additional passages on C6/36 were performed before concluding that DENV did not replicate.
Our positive controls consisted of urine and saliva specimens obtained from healthy individuals as well as VTM spiked with infectious virus at a final concentration of 3 to 7 log10 cDNA copies/ml. Spiked urine controls were dialyzed and concentrated as for the patient’s urine samples.

Andries A.C., Duong V., Ly S., Cappelle J., Kim K.S., Lorn Try P., Ros S., Ong S., Huy R., Horwood P., Flamand M., Sakuntabhai A., Tarantola A, & Buchy P. (2015). Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens. PLoS Neglected Tropical Diseases, 9(9), e0004100.

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Quantifying Dopamine Deposits in Tick Hemocytes

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Four experimental groups with 25 ticks each were evaluated: C, D, M, and DM. The females were inoculated between the scutum and capitulum using a microinjector (Drummond, Broomall, PA, USA). The hemolymph was collected [13 (link)] in 500 μL L-15 Leibovitz medium (Sigma-Aldrich, St. Louis, MO, USA), 24 hpi. The hemocytes were quantified in a Neubauer chamber, adjusted to 2 × 10⁴ cells on a circular coverslip, and placed in a 24-well culture plate (Kasvi, São José dos Pinhais, Brazil). The hemocytes were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 30 min and washed in PBS three times. Hemocytes were incubated with primary anti-dopamine antibody (ab6427; Abcam, Cambridge, UK) for 72 h and with the secondary antibody [Goat anti-rabbit Alexa Fluor 594 (red); Invitrogen, Waltham, MA, USA)] for one hour at room temperature. The hemocytes’ nuclei were stained with DAPI (blue) at room temperature, and the hemocytes were observed under a fluorescence microscope (BX 51; Olympus, Tokyo, Japan) according to the protocol adapted described by Wu et al. [19 (link)]. DA deposits were quantified using ImageJ 1.52a software (National Institute of Health, Bethesda, MD, USA), and the results are presented as the percentage of marked area. The experiment was performed twice.

Corrêa T.A., Fiorotti J., Mesquita E., Meirelles L.N., Camargo M.G., Coutinho-Rodrigues C.J., Marciano A.F., Bittencourt V.R, & Golo P.S. (2021). How Dopamine Influences Survival and Cellular Immune Response of Rhipicephalus microplus Inoculated with Metarhizium anisopliae. Journal of Fungi, 7(11), 950.

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Ethanol Inactivation of Orthonairovirus

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Hazara orthonairovirus (HAZV) was used as a model orthonairovirus for this study. HAZV was grown SW-13 cells with L-15 (Leibovitz) medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 100 U/ml penicillin (Sigma-Aldrich), 0.1 mg/ml streptomycin (Sigma-Aldrich) and 2% fetal bovine serum. The virus was harvested 48 h post infection and the tissue culture infectious dose (TCID₅₀) was calculated by the Spearman-Karber method. In order to create conditions similar to blood-fed infected ticks from the field and thus account for the possible influence of host blood, the virus-containing cell culture supernatant (10%) was mixed with bovine serum (80%) as well as bovine EDTA (10%). In this mixture, the virus was exposed to concentrations of 0–60% ethanol using a dilution series. The blood/virus mixture was eventually incubated under various temperature conditions (− 20 °C, 4 °C or room temperature) for four time periods (0, 3, 9, 24 h). Afterwards, the residual virus infectivity was determined by titration on SW-13 cells (6-well plates). Due to the cytotoxicity of ethanol, the virus cultivation at 6-well plates was conducted in an additional 1:20 dilution using the same media as described above. All samples were cultured in duplicate including positive and negative controls.

Schulz A., Methling K., Lalk M., Eisenbarth A., Keller M, & Groschup M.H. (2021). Ethanol inactivation of orthonairoviruses in ixodid ticks. Experimental & Applied Acarology, 85(1), 75-81.

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Rainbow Trout Liver Cell Culture

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The rainbow trout liver cell line (RTL-W1)
[82 (link)] was grown in L15-Leibovitz medium (Sigma-Aldrich) supplemented with 9% fetal bovine serum (FBS, Biowest, Logan, UT, USA) and penicillin/streptomycin (10,000 E/mL; 10,000 μg/mL in 0.9% NaCl, Sigma-Aldrich) in 75-cm² flasks (Techno Plastic Products (TPP), Trasadingen, Switzerland) at 20°C in darkness according to the protocol detailed in Klee et al.
[83 ].

Simon A., Maletz S.X., Hollert H., Schäffer A, & Maes H.M. (2014). Effects of multiwalled carbon nanotubes and triclocarban on several eukaryotic cell lines: elucidating cytotoxicity, endocrine disruption, and reactive oxygen species generation. Nanoscale Research Letters, 9(1), 396.

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Isolation of Trigeminal Ganglion Neurons

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Trigeminal ganglion tissues were collected from postnatal day 2–8 mice of both sex in L15 Leibovitz medium (Sigma) and treated with 2 mg/mL collagenase type II (Worthington) and BSA (Euromedex) for 2 hours, followed by 2.5 mg/mL trypsin (Sigma) for 15 min. Neurons were dissociated in DMEM/F12 medium (Gibco) by triturating with fire-polished and coated glass pipettes and seeded on poly-L-lysine (Sigma) coated coverslips. The DMEM-based culture medium contained 10% fetal bovine serum (Dutscher) and 2 mM Glutamine (Gibco).

Ávalos Prado P., Landra-Willm A., Verkest C., Ribera A., Chassot A.A., Baron A, & Sandoz G. (2021). TREK channel activation suppresses migraine pain phenotype. iScience, 24(9), 102961.

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Transgender Men Ovarian Collection

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Following bilateral oophorectomy of transgender men, sibling ovaries were collected in prewarmed (37 o C) or cold collection (on ice) medium (L-15 Leibovitz medium, Sigma) supplemented with 0.45% human serum albumin (Red Cross, Belgium). Ovaries were collected immediately after isolation from the surgical room of Ghent University Hospital and transferred within 10 minutes to the laboratory, where ovarian weight was recorded (Supplementary Table SII). Ovaries collected in warm medium were manipulated first, whilst ovaries collected in cold medium stayed on ice for 2-3hrs following collection. For seven of the patients, both ovaries were collected on ice.

Christodoulaki A., He H., Zhou M., Cardona Barberán A., De Roo C., Chuva De Sousa Lopes S.M., Baetens M., Menten B., Van Soom A., De Sutter P., Weyers S., Boel A., Stoop D, & Heindryckx B. (2023). Characterization of ovarian tissue oocytes from transgender men reveals poor calcium release and embryo development, which might be overcome by spindle transfer. Human reproduction (Oxford, England), 38(6).

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Transfection of Cells with pEGFP-actin

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The plasmid used for transfection was pEGFP-actin (Clontech). Transfection was performed by mixing 100ml OPTIMEM, 6ml Fugene 6 (Roche) and 1mg plasmid DNA for 30min and adding it to the cells in L-15 Leibovitz medium (Sigma) for 5h. Then the medium was changed to START medium again and the transfected cells were re-plated onto coverslips as described above.

Mueller J., Szep G., Nemethova M., de Vries I., Lieber A.D., Winkler C., Kruse K., Small J.V., Schmeiser C., Keren K., Hauschild R, & Sixt M. (2017). Load Adaptation of Lamellipodial Actin Networks. Cell, 171(1).

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Viral Growth Kinetics in E-11 Cells

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E-11 cells were grown in 5 mL culture flasks with Leibovitz medium (L-15) (Sigma-Aldrich), supplemented with 10% FCS, L-Glutamine (2 mM) and antibiotics (100 IU/mL penicillin, 100 μg/mL streptomycin and 0,25 μg/mL Amphotericin B). Cell monolayers were infected in three replicates with each of the isolates at a multiplicity of infection (MOI) of 1.0. After 1 h adsorption at 25 °C, all the inocula were brought to the same volume with L-15 medium without growth factor (FCS). Inoculated monolayers were then incubated at 15, 20, 25 and 30 °C and checked regularly for CPE. A volume of 700 μL was sampled from each flask at 0 (T₀), 20 (T₁), 30 (T₂), 45 (T₃), 55 (T₄), 69 (T₅), 79 (T₆), 93 (T₇), 117 (T₈), 141 (T₉) and 165 (T₁₀) hours post infection (hpi), and subsequently replaced with an equal amount of medium. Collected cell culture supernatants were subjected to viral titration in E-11 monolayers by endpoint dilutions assays. Viral titres were calculated according to the Spearman-Karber formula [40 ]. Finally, average titres expressed as TCID₅₀/mL were calculated among replicates, and were used for developing growth curves for each incubation temperature.

Panzarin V., Cappellozza E., Mancin M., Milani A., Toffan A., Terregino C, & Cattoli G. (2014). In vitro study of the replication capacity of the RGNNV and the SJNNV betanodavirus genotypes and their natural reassortants in response to temperature. Veterinary Research, 45(1), 56.

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Betanodavirus Genetic Variants Characterization

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On the basis of previous phylogenetic analysis of partial RNA1 and RNA2 sequences [23 (link),36 (link)], four betanodavirus isolates representative of the RGNNV (283.2009), SJNNV (484.2.2009), RGNNV/SJNNV (367.2.2005) and SJNNV/RGNNV (389/I96) genetic variants were selected for further genetic and phenotypic characterization (Table 1). All but one of the selected viruses (283.2009; 367.2.2005; 389/I96) were originally isolated from the same fish species (sea bass, Dicentrarchus labrax). However, strain 484.2.2009 isolated from a Senegalese sole (Solea senegalensis) was also included in the selection, because to date no SJNNV infection has been reported in sea bass.
Betanodavirus isolates were propagated in E-11 cell monolayers (ECACC no. 01110916; [12 (link)]) incubated at 25 °C in Leibovitz medium (L-15) (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% FCS, L-Glutamine (2 mM) and antibiotics (100 IU/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B) [37 (link)]. Inoculated cell cultures were checked daily for cytopathic effect (CPE). Supernatants were collected upon disruption of cell monolayers, clarified by centrifugation at 4000 × g for 15 min at 4 °C, and subsequently subjected to further investigations.

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Betanodavirus Isolates Experimental Challenge

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Four isolates of Betanodavirus with diverse genomes (283.2009: RGNNV; 367.2.2005: RGNNV/ SJNNV; 389/I96: SJNNV/RGNNV; 484.2.2009: SJNNV) were used for the experimental challenge of sea bass juveniles (Table 1). They were fully sequenced, and their growth kinetics on cell culture in response to temperature had already been thoroughly investigated by Panzarin et al. (2014) (link).
Viral strains were multiplied in E-11 cells (European Collection of Authenticated Cell Cultures, ECACC no. 01110916; Iwamoto et al. 2000) (link) at 25°C, with Leibovitz medium (L-15) (Sigma) containing 10% fetal bovine serum (FBS), L-glutamine (2 mM) and antibiotics (100 IU ml -1 penicillin, 100 µg ml -1 streptomycin and 0.25 µg ml -1 amphotericin B). Infected monolayers were che cked regularly for the presence of cytopathic effect (CPE), and cell supernatant was collected upon disruption of cell monolayers. The supernatant was clarified by centrifugation at 4000 × g (15 min at 4°C) and then titrated. Titration was performed (in quadruplicate) on 1 d old E-11 cells seeded in 96-well plates (Corning) with L-15, supplemented with 10% FBS, L-glutamine (2 mM) and antibiotics. Plates were observed after 10 d for CPE appearance, and viral titre was calculated according to the Spearman and Karber formula (Finney 1978) . All isolates used in this study were at seventh passage on E-11 cells.

Toffan A., Panzarin V., Toson M., Cecchettin K, & Pascoli F. (2016). Water temperature affects pathogenicity of different betanodavirus genotypes in experimentally challenged Dicentrarchus labrax. Diseases of aquatic organisms, 119(3).

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