HEK 293 and A549 (human lung epithelial) cells were cultured in DMEM (Sigma-Aldrich, St. Louis, USA) supplemented with 10% FCS (10% FCS/DMEM) and antibiotics at 37 °C in 5% CO2. Madin–Darby canine kidney (MDCK) cells were cultured in Eagle’s MEM (GIBCO) with 5% NCS at 37 °C in 5% CO2. Influenza A/WSN/33 virus (WSN; H1N1) was generated by use of reverse genetics, as described previously (Neumann et al., 1999 (link)) and propagated in MDCK cells. Virus was titrated by plaque assay in MDCK cells (Tobita et al., 1975 (link)). The PB2-knockout virus possessing the Renilla luciferase gene (PB2-KO/Rluc virus) was generated with pPolIPB2(120)Rluc(120) (encoding Renilla luciferase flanked by 120 N- and C-terminal nucleotides derived from the PB2 segment) and plasmids encoding the remaining seven viral RNA segments, and propagated and titrated in MDCK cells stably expressing the PB2 protein as described previously (Ozawa et al., 2011 (link)).
Eagle s mem
Manufactured by Thermo Fisher Scientific
Sourced in United States
Eagle's Minimum Essential Medium (Eagle's MEM) is a cell culture medium formulated to support the growth and maintenance of various cell types. It provides a balanced combination of amino acids, vitamins, salts, and other essential nutrients required for cell proliferation. Eagle's MEM is a widely used basal medium in cell biology research and applications.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Complete dmem media, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Avantor (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Digitoxin, by Merck Group (1 mentions)
Celltiter glo, by Promega (1 mentions)
Ham s f 12k, by Fujifilm (1 mentions)
Dmem medium, by PAN Biotech (1 mentions)
36 protocols using eagle s mem
1
Influenza A virus propagation in cells
2
Characterization of HSV-1 Infection in Keratinocytes
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The wild-type HSV-1 virus Syn17+ [88 (link)] was used throughout the study, and the virus titers were determined by plaque titration on Green monkey kidney (GMK, obtained from the Swedish Institute for Infectious Disease Control, Stockholm) cells as previously described [89 (link)]. HSV-1 Syn17+ virus was cultivated in HaCaT wild type keratinocytes or GMK cells depending on downstream application and the titers were determined as mentioned above. Diploid human embryonic lung fibroblasts [70 (link)] (HEL, obtained from the cell culture collection at the Sahlgrenska University Hospital, department of Clinical Microbiology, Gothenburg) at a low passage level were cultivated in Eagle’s MEM (Gibco, Life Technologies) with 10% FCS (Sigma), 100 IU/mL penicillin, 100 μg/mL streptomycin (Gibco, Life Technologies) and 2 mM L-glutamine. HaCaT wild type [90 (link)] and HaCaT COSMC-/- [56 ] keratinocytes were grown in DMEM (Gibco, Life Technologies), supplemented with 10% FCS (HyClone), 100 IU/mL penicillin and 100 μg/mL streptomycin (Gibco, Life Technologies). HaCaT COSMC-/- clone D5 harbors a 10 bp deletion at the zink finger nuclease target site of COSMC gene, whereas clone E5 harbors a combined 12 bp deletion and a 2 bp insertion. Both genetic alerations result in introduction of STOP codons due to frameshift mutations [56 ].
3
Influenza A/WSN/33 virus generation
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Influenza A/WSN/33 virus (WSN) (H1N1) was generated using reverse genetics (66 (link)). HEK293 cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FCS (10% FCS/DMEM) and antibiotics at 37°C in 5% CO2. Virus plaque titers were determined by plaque assay in Madin-Darby canine kidney (MDCK) cells. MDCK cells were cultured in Eagle’s MEM (Gibco) with 5% NCS at 37°C in 5% CO2.
4
Cell Viability Assay with HepG2 Cells
5
In vitro Culture and Viral Propagation Protocol
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A549 (human lung epithelial) cells were cultured in Ham’s F-12K (Wako) supplemented with 10% FCS at 37°C in 5% CO2. Madin-Darby Canine Kidney (MDCK) cells were cultured in Eagle’s MEM (GIBCO) with 5% NCS at 37°C in 5% CO2. A/WSN/1933 (H1N1) was propagated in MDCK cells and titrated by plaque assay in MDCK cells (Tobita et al., 1975 (link)). The PB2-knockout virus encoding Renilla luciferase (PB2-KO/Rluc virus) was propagated and titrated in MDCK cells stably expressing the PB2 protein as described previously (Ozawa et al., 2011 (link)). All experiments were performed under biosafety level 2 (BSL2) conditions.
6
Neutralization Assay for CA16 Virus
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Neutralization tests were performed in RD cells using a TCID50-reduction assay [19 (link), 42 (link)]. Serum samples were incubated at 56°C for 30 min. Duplicate serum samples were diluted in Eagle’s MEM (Gibco) in a series ranging from 1:8 to 1:1024 dilutions. A total of 50 μl of 100 tissue culture infective doses (TCID50) of CA16 virus was mixed with 50 μl of the appropriate serum dilution and incubated at 37°C for two hours in the presence of CO2, followed by the addition of 100 μl of an RD cell suspension (1 x 105 cells per 0.1 ml). Infected cells and controls were incubated at 37°C in 5.0% CO2 and observed with an inverted microscope daily for seven days. The highest dilution that prevented the development of cytopathic effects in 50.0% of the wells was considered the antibody titer of the sample. A virus back-titration was also performed to determine the virus titer and this serve as a no-serum control.
7
Cell Culture Protocols for Diverse Cell Lines
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The human keratinocyte cell line (HaCaT), COS7 and ovarian adenocarcinoma cell lines were cultured in DMEM medium (PAN Biotech No. P04–03550) with high glucose (4,5 g/dL), penicillin/streptomycin, glutamate and 5% fetal bovine serum. The ovarian adenocarcinoma cell lines were a kind gift from Prof. Baki Akgül (Institute of Virology, Medical Faculty, University of Cologne).34,35 (link) Cells were grown in a humidified atmosphere and 5% CO2. Subcutaneous fibroblasts from a patient suffering from EDMD5 were a gift from Prof. Wehnert (Institute of Human Genetics and Interfaculty Institute of Genetics and Functional Genomics, University Greifswald).24 (link) The fibroblasts (Passage 13–17) were cultivated in Eagle's MEM (Gibco, No. 31095–029), 20% fetal bovine serum, 7.5% Bicarbonate, 1 M HEPES, penicillin-streptomycin and glutamate.
8
Pituitary RPD Isolation and Incubation
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Procedures for pituitary RPD isolation and incubations have been described previously (30 (link), 32 (link)). The pituitary was removed and the RPD was dissected and placed individually in wells of a 96-well plate containing Modified Kreb's bicarbonate Ringer (320 mOsmolal) solution with addition of glucose (5 mM), L-glutamine (2.05 mM), and Eagles MEM (Gibco, Grand Island, NY) whereby pH of the media was adjusted to 7.4. Tissues were incubated at 24°C in a humidified environment containing 95%O2/5%CO2 at 60 revolutions per minute. Following a 2-h preincubation, the media was replaced with fresh control or experimental media containing recombinant tilapia Leptin A (rtLepA, dosages indicated in figures and legends), the dominant leptin paralog in teleost fishes (30 (link)), and/or pharmacological agents at concentrations and time periods provided in the figures (33 (link)). Media was removed and frozen at −20°C for subsequent lactate measurement. Tissues were removed and placed in either RNALater at 4°C for assessment of gene expression or in phosphofructokinase (PFK) assay buffer (Sigma), snap-frozen in liquid nitrogen then placed at −20°C for later Western blot analyses and PFK activity determinations.
9
Chromosomal Aberration Assay of MWCNTs
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Seven different MWCNTs (CTa, CTb, CTc, CTd, CTe, CTf, and CTg) were used for chromosomal aberration assays. CTa (MWNT-7) and CTb were obtained from Hodogaya Chemical Co. Ltd. (Tokyo, Japan); CTc, CTd, and CTg were obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan); CTe was obtained from Showa Denko K.K. (Tokyo, Japan); and CTf was made by shattering CTe in an agate mortar. Eagle's MEM was purchased from GIBCO (Invitrogen Cell Culture, CA, USA), and calf serum was purchased from SAFC Biosciences (Kansas City, USA). Dimethyl sulfoxide (DMSO), which was used as a solvent, and Mitomycin C (MMC), which was used as a positive control, were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
10
Vero and BHK-21 Cell Culture and Antibody Panel
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Vero cells (African green monkey kidney cells originally acquired from the American Type Culture Collection ∼40 years ago) were propagated in Eagles MEM (Gibco) supplemented with 7% FBS, 100 g/mL penicillin/streptomycin (Gibco), 2 mM L-glutamine (Gibco). BHK-21 cells were grown 10% FBS, 100 g/mL penicillin/streptomycin, 2 mM L-glutamine, 7% tryptose phosphate broth solution (Sigma), and 0.56% glucose (wt/vol). The following anti-mouse antibodies were purchased from BD Biosciences: PE CD4 (RM4.5), PE IFNγ, PerCP-Cy5.5 CD3 (145-2c11), PE Ly6G (1A8), and PerCP-Cy5.5 NK1.1 (145-2c11). The following anti-mouse antibodies were purchased from eBioscience: FITC TNFα (MP6-X722), PE SiglecH (440C), PerCP-Cy5.5 CD11c (N418), PerCP-Cy5.5 Ly6c (HK1.4), e450 and APC CD8a (53–6.7), e450 B220 (RA3-6B2), e450 and PE-Cy7 CD90.2 (53–2.1), e450 and PE-Cy7 CD19 (1D3), PE-Cy7 CD3 (145-2c11), PE-Cy7 CD21/CD35, PE-Cy7 CD11b (M1/70), APC and APC-Cy7 CD45 (30F11), APC CD31 (390), and APC PDCA-1 (927). The following anti-mouse antibodies were purchased from BioLegend: PE gp38 (8.1.1), PE-Cy7 CD8a (53–6.7), and APC F4/80 (BM8). Anti-LCMV NP antibody (VL-4) was purchased from BioXcell and conjugated to Alexa Fluor 488 using antibody labeling kit from Life Technologies.
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