The RAW264.7 cell line was obtained from the European Collection of Cell Culture. Cells were grown at 37 °C and 5% (v/v) CO2in tissue culture flasks (Nagle Nunc. International, Hereford, UK) containing DMEM medium (Lonza Group Ltd, Braine-l’Alleud, Belgium), added with 1% (w/v) L-glutamine (Invitrogen, Paisley, UK), 10% (v/v) heat inactivated foetal calf serum (Biosera, Ringmer, UK), and 2% (w/v) penicillin/streptomycin antibiotics (Invitrogen Ltd, Paisley, UK). For cytokine stimulation, cells were gently and mechanically detached from bottles, and 2*105 cells/well were seeded in a 96-well flat-bottom plate. The cells were left to adhere overnight at 37 °C and 5% (v/v) CO2. 100 µl fresh medium was added to the wells and then stimulated with 2*105 heat-killed yeast cells for 24 h at 37 °C and 5% (v/v) CO2. For blocking with glucan-phosphate-treated, cells were incubated with 10.0 µg of the glucan-phosphate for 2 h at 37 °C and 5% (v/v) CO2 before challenge with fungal cells. To analyze the effect of temperature-induced cell wall changes in wild type strains, the RAW macrophages were similarly challenged with thimerosal killed fungal cells exposed to different temperatures, as described in the methods section. Upon stimulation, supernatants were collected as described above and used to quantify murine TNFα by ELISA using kits from R&D.
Foetal calf serum
Manufactured by Biosera
Sourced in United Kingdom, France
Foetal calf serum is a cell culture supplement derived from the blood of foetal calves. It is used to provide essential nutrients, growth factors, and other components required for the in vitro cultivation of a wide range of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Merck Group (6 mentions)
L glutamine, by Merck Group (4 mentions)
Streptomycin, by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Merck Group (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Hiv 1 p24 elisa, by PerkinElmer (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Lonza (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Lonza (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Auranofin, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Pr 619, by LifeSensors (1 mentions)
Puromycin, by Santa Cruz Biotechnology (1 mentions)
Bafilomycin a1, by Enzo Life Sciences (1 mentions)
Z vad, by Merck Group (1 mentions)
24 protocols using foetal calf serum
1
Assessing Macrophage Cytokine Response to Fungal Stimuli
2
Generation of Viral-Like Particles and Stable Cell Lines
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HEK 293T, HeLa and D17 cells (Bishop laboratory cell stocks) were maintained in DMEM (Thermo Fisher Scientific), supplemented with 10% heat-inactivated foetal calf serum (Biosera) and 1% penicillin-streptomycin (Sigma). The cells were stored in a humidified incubator at 37°C and 5% CO2.
VLPs were made by co-transfecting 293T cells with plasmids encoding VSV-G (pczVSV-G), Mo-MLV Gag-Pol and the LacZ reporter gene (pczLTR-LacZ), in an equimolar ratio. Approximately 16 h after transfection the cells were treated with 10 mM sodium butyrate for 6 h to promote transcription. VLP-containing culture supernatants were harvested 48 h post-transfection and filtered to remove cellular debris. Viral titres were quantified using a modified ELISA for reverse transcriptase activity (Cavidi). Transduction vectors for stable cell line generation were produced by co-transfecting 293T cells with plasmids encoding VSV-G (pczVSV-G), Mo-MLV Gag-Pol (KB4) and GST-p12, as described above. HeLa cells were transduced with these VLPs by spinoculation (1600 g, 2 h, 16°C) in the presence of 4 μg/ml polybrene (Sigma). From approximately 72 h after infection, cells were passaged in media containing 0.5 μg/ml puromycin (Thermo Fisher Scientific) to select for transduction.
VLPs were made by co-transfecting 293T cells with plasmids encoding VSV-G (pczVSV-G), Mo-MLV Gag-Pol and the LacZ reporter gene (pczLTR-LacZ), in an equimolar ratio. Approximately 16 h after transfection the cells were treated with 10 mM sodium butyrate for 6 h to promote transcription. VLP-containing culture supernatants were harvested 48 h post-transfection and filtered to remove cellular debris. Viral titres were quantified using a modified ELISA for reverse transcriptase activity (Cavidi). Transduction vectors for stable cell line generation were produced by co-transfecting 293T cells with plasmids encoding VSV-G (pczVSV-G), Mo-MLV Gag-Pol (KB4) and GST-p12, as described above. HeLa cells were transduced with these VLPs by spinoculation (1600 g, 2 h, 16°C) in the presence of 4 μg/ml polybrene (Sigma). From approximately 72 h after infection, cells were passaged in media containing 0.5 μg/ml puromycin (Thermo Fisher Scientific) to select for transduction.
3
Characterization of p62 Knockout MEFs
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HeLa and HEK293E cells were obtained from ECACC (European Collection of Cell Cultures). p62 knockout (p62−/−) and wild-type (p62+/+) mouse embryonic fibroblasts (MEFs) were kindly provided by Eiji Warabi of the University of Tsukuba. HEK293FT lentivirus packaging cells were from Invitrogen (Paisley, UK). Cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma #D6546) supplemented with 10% heat-inactivated FCS (Foetal Calf Serum, Biosera), 5% penicillin/streptomycin (Invitrogen) and 2mM l -glutamine (Sigma) in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were treated with puromycin (Santa Cruz Biotechnology), staurosporine (Sigma), z-VAD (Sigma), H2O2 (Sigma; the H2O2 stock was replaced every 2 weeks for the duration of the project), PR-619 (LifeSensors), curcumin (Sigma), auranofin (Sigma), N-acetylcysteine (Sigma), bafilomycin A1 (Enzo Life Sciences), chloroquine (Sigma), cycloheximide (Sigma) and retinoic acid (as an inhibitor of Nrf247 (link)) at different concentration and time-points as indicated. Medium was switched to serum-free DMEM for the duration of the treatments.
4
Synaptic Vesicle Recycling Dynamics
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FM1‐43, tetramethylrhodamine‐dextran (TMR‐dextran), penicillin/streptomycin, phosphate‐buffered salts, and Minimal Essential Medium were obtained from Invitrogen. Foetal calf serum was from Biosera. The dynamin I phospho‐specific Ser‐774 antibody was from AbD Serotec, the synaptophysin antibody was from Synaptic Systems and the synaptobrevin II antibody was from Nventa Biopharmaceuticals. Glutaraldehyde and osmium tetroxide were from Agar Scientific. Tetanus toxin (TnTx), BAPTA‐AM (1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid) and EGTA‐AM were from Calbiochem. Fura‐2 was from Anaspec. Synaptophysin‐pHluorin (sypHy) was a gift from Prof. Leon Lagnado (University of Sussex). All other reagents were from Sigma.
5
Maintenance of Diverse Cell Lines
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293T, TE671, D17 and M. dunni cells (Bishop laboratory cell stocks) and U20S and U/R cells (Bacharach laboratory cell stocks) were maintained in DMEM (Invitrogen) supplemented with 10% heat inactivated foetal calf serum (Biosera) and 1% penicillin/streptomycin (Sigma), in a humidified incubator at 37°C and 5% CO2. U/R cells which stably express mCAT-1 [12] (link) were maintained in the presence of 100 ug/ml Zeocin (Invitrogen).
6
Generating and Titrating Retroviral Vectors
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293T and TE671 cells were maintained in DMEM (Invitrogen) and U937 cells in RPMI +[L]-Glutamine (GIBCO), each supplemented with 10% heat inactivated foetal calf serum (Biosera) and 1% penicillin/streptomycin (Sigma). U937 cells stably expressing SAMHD1 variants were prepared by transduction with MoMLV-based Puromycin-expressing VLPs, followed by selection with puromycin at 10 μg/mL. MoMLV-based YFP-expressing VLPs were made by cotransfecting 293T cells with pVSV-G (gifted from D. Lindemann), pKB4 [37 (link)] and pLGateway_SAMHD1IRESYFP (wild-type or mutants), harvesting 48 hr post-transfection. HIV-1GFP was produced by cotransfection of pVSV-G, p8.91 and pCSGW [38 (link)]. MoMLV-based Puromycin-expressing VLPs were made by cotransfection of pKB4, pVSV-G and pCMS28_SAMHD1 (wild-type or mutants). VLPs were titred on TE671 or 293T cells for normalisation prior to infection. Viruses with mutations in Reverse Transcriptase were normalised by HIV-1 p24 ELISA (Perkin Elmer).
7
Breast Cancer Cell Lines for HER2 Studies
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Four human breast cancer cell lines were used: SKBR3, MCF-7, BT474 and ZR-175. SKBR-3 is a human epithelial cell line that overexpresses the HER2 receptor29 (link) and has been widely used for HER2 expression studies. BT474 is also a HER2 overexpressing cell line. Both MCF-7 and ZR-175 are known to be HER2 negative. SKBR-3 cells were cultured in McCoy’s 5 A medium with 2 mM L-glutamine (Lonza, Slough, UK) supplemented with 10% (v/v) foetal calf serum (FCS; Biosera, Ringmer, UK). The remaining cell lines were grown in DMEM (Lonza, Slough, UK) supplemented with 10% (v/v) FCS. All cells were incubated at 37 °C in humidified atmosphere in a 5% v/v CO2 incubator. The cells were passaged at the split-ratio recommended by the ATCC and were seeded into 6-well plates using standard cell culture techniques. All the experiments were performed within a total of ten passages.
8
Ewing Sarcoma Cell Line Cultivation
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Cells were obtained from ATCC or different partner institutes of EuroBoNeT (Table S1 ) [37] (link). All EWSR1 translocation confirmed Ewing sarcoma (ES) cell lines used in this study were grown in RPMI 1640 (PAA Laboratories GmbH, Austria) supplemented with 1% Penicillin-Streptomycin (PAA Laboratories GmbH, Austria) and 10% Foetal Calf Serum (Biosera, UK). Three cell lines (STA-ET 2.1, STA-ET10, WE-68) needed to be cultivated in gelatine-coated culture flasks to allow cells to attach. For growth factor experiments cell lines were grown on coverslips (d = 13 mm) in 24-well plates (Costar, USA) with 4×104 cells per well. Poorly attaching cell lines (e.g. STA-ET 2.1, STA-ET10, WE-68) were seeded on either Matrigel or gelatine coated coverslips (growth factor reduced, BD Biosciences, UK). After adaption for 2 days, cells were serum-starved in RPMI 1640 supplemented with 1% Penicillin-Streptomycin for 24 hr and treated with IGF2 (50 ng ml−1, R&D systems) for 1 hr at 37°C. Finally cells were fixed in 4% (v/v) formaldehyde for 15 min at room temperature (RT).
9
Generating Viral-Like Particles for Cell Transduction
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293 T cells were maintained in DMEM (Invitrogen) and U937 and Jurkat T-cells were cultured in RPMI containing [L]-Glutamine (ThermoFisher) at 37 °C in 5% CO2. Media were supplemented with 10% heat-inactivated foetal calf serum (Biosera) and 1% penicillin/streptomycin (Sigma). MLV-based VLPs used to transduce cells with SAMHD1 were produced by co-transfecting 293 T cells with pVSV-G, pKB4 and pLGateway_SAMHD1IRESYFP (either WT or SAMHD1 mutant). HIV-1-GFP VLPs were produced by co-transfecting 293 T cells with pCSGW, pVSV-G, and p8.91 (either WT or RT mutant). SIVmac VLPs containing or lacking Vpx were produced by co-transfecting 293 T cells with pVSV-G and either pSIV3+ (for Vpx +) or pSIV3+vpx- (for Vpx-). VLP-containing cell culture supernatants were harvested 48 h post-transfection and titred on TE671 or 293 T cells for normalisation prior to infection. VLPs with mutations in Reverse Transcriptase were normalised by HIV-1 p24 ELISA (Perkin Elmer).
10
Culturing tsA-201 Cells for Experiments
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tsA‐201 cells, a modified HEK‐293 cell line, were maintained in 5% CO2, in a humidified incubator at 37°C, in growth media containing Minimum Essential Media (MEM) (with Earle's, without L‐glutamine), 10% foetal calf serum (Biosera, Sussex, UK), 1% nonessential amino acids (Gibco, Paisley, UK), 1% penicillin (10 000 U mL−1) and streptomycin (10 mg mL−1) (Sigma, Dorset, UK). When the cells were 90% confluent, they were split and plated on 13 mm glass coverslips coated with poly‐L‐lysine (0.1 mg mL−1, Sigma).
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