For bone marrow-derived MC (BMMC) culture, WT bone marrow was obtained from WT C57BF/6 mice in ice-cold Hank's Balanced Salt Solution (HBSS, Gibco), and cultured in complete RPMI containing 10% fetal bovine serum (FBS) (Hyclone), 1 mM nonessential amino acids, 25 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 1× Antibiotic-Antimycotic (all reagents from Gibco), 10 ng/ml SCF and 5 ng/ml IL-3 (BioLegend) for 8 to 12 weeks. For BMDC culture, bone marrow was obtained from WT C57BL/6 or CD301b-DTR-GFP mice and cultured in RPMI media containing 10% FBS, 1× GlutaMAX® (Gibco), 20 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF) (BioLegend), or 20 ng/ml GM-CSF and 50 ng/ml IL-4 for 6-7 days. For bone marrow-derived macrophage (BMM) culture, the same procedures were followed replacing the growth factors with 20 ng/ml macrophage colony-stimulating factor (M-CSF). The rat MC RBL-2H3 cells (ATCC) were cultured in minimum essential medium (MEM) medium (Gibco) containing 15% FBS and antibiotics. JAWSII cells (ATCC) were maintained in MEM α medium (Gibco) supplemented with 20% FBS, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin. All cells were cultured at 37 °C in a humidified water-jacketed incubator under 5% CO2 / 95% air atmosphere.
Jawsii cells
Manufactured by American Type Culture Collection
Sourced in United States
JAWSII cells are a cell line derived from human osteosarcoma tissue. They are adherent cells that can be used for various in vitro studies.
Automatically generated - may contain errors
Lab products found in correlation
Kaluza analysis software, by Beckman Coulter (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Interleukin 3 (il 3), by BioLegend (1 mentions)
Stem cell factor (scf), by BioLegend (1 mentions)
Gm csf, by BioLegend (1 mentions)
α mem medium, by Thermo Fisher Scientific (1 mentions)
Rbl 2h3 cells, by American Type Culture Collection (1 mentions)
Interleukin 4 (il 4), by BioLegend (1 mentions)
Granulocyte macrophage colony stimulating factor gm csf, by BioLegend (1 mentions)
Mem medium, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Mouse igg2a, by BioLegend (1 mentions)
Alexa fluor 647 goat anti rabbit igg h l highly cross adsorbed secondary antibody, by Thermo Fisher Scientific (1 mentions)
Raw blue cells, by InvivoGen (1 mentions)
Mc38 cells, by Kerafast (1 mentions)
B16f10 cells, by American Type Culture Collection (1 mentions)
J774 cells, by American Type Culture Collection (1 mentions)
Calu 3 cells, by American Type Culture Collection (1 mentions)
9 protocols using jawsii cells
1
Isolation and Culture of Bone Marrow-Derived Cells
2
Quantifying SARS-CoV-2 S1 Protein Binding
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JAWSII cells (ATCC) were seeded onto a 96 well hanging drop plate (Sigma-Aldrich) at 5 x 104 cells/well. EDV only, EDV-αGC, EDV-CONTROL, EDV-COVID and EDV-COVID-αGC were co-incubated with the cells at 1x109 EDVs per well. Untreated JAWSII cells were used as controls. The samples were cultured at 37°C with 5% CO2 for 48 h before being collected and co-stained with PE anti-mouse αGC:CD1d complex antibody (ThermoFisher, 1:2000) and SARS-CoV-2 S1 protein polyclonal primary antibody (GeneTex, 1:2000) at room temperature for 30 min in the dark. The samples were then stained with Alexa Fluor 647 goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (ThermoFisher, 1:1000) at 4°C for a further 20 min and analysed using a Gallios flow cytometer (Beckman Coulter). Mouse IgG2a (Cat. #400214, Biolegend) and rabbit IgG (Abcam) were used as isotype controls. DAPI was used to differentiate live/dead cells and single stained samples were used to generate compensation. The samples were analysed using the Kaluza analysis software (V2.1, Beckman Coulter).
3
EDV-COVID-αGC Stimulation of JAWSII Cells
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JAWSII cells (ATCC) were treated with EDV-COVID-αGC in a 96-well Perfecta3D hanging drop plate (Cat. #HDP1385, Sigma-Aldrich) at 1x109 EDV-COVID-αGC per cell. JAWSII cells treated with 2 µg/mL αGC (Advanced Molecular Technologies) served as a positive control. The cultures were then incubated for 24 h at 37°C with 5% CO2 and cells were collected and stained with a PE conjugated CD1d: αGC complex antibody (Cat. #12-2019-82, ThermoFisher, 1:2000) and analyzed using a Gallios flow cytometer (Beckman). The results were analyzed using Kaluza Analysis software (V.2.1, Beckman).
4
Cell Culture Conditions for Diverse Murine Cell Lines
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B16F10 cells were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 units/mL aqueous Penicillin G sodium and 100 µg/mL streptomycin (Pen/Strep) at 37 °C in 5% CO2 humidified air. JAWSII cells were purchased from ATCC and cultured in alpha minimum essential medium with ribonucleosides, deoxyribonucleosides, 4 mM l -glutamine, 1 mM sodium pyruvate, and 5 ng/mL murine GMCSF, 80%; FBS, 20%. MC38 cells were purchased from Kerafast, Inc. and cultured in DMEM containing 10% FBS, 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 50 µg/mL gentamycin sulfate, Pen/Strep. TLR2 reporter cell line RAW-Blue cells were purchased from Invivogen and cultured in DMEM containing 10% FBS, 100 mg/mL Normocin, 2 mM glutamine, and 200 µg/mL Zeocin.
5
Culturing Murine Cell Lines
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Murine dendritic JAWSII cells (ATCC, Manassas, VA, USA) were maintained in MEM α, nucleosides, with no ascorbic acid, 1 mM sodium pyruvate and 5 ng/ml GM-CSF Recombinant Mouse Protein cultured at 37 °C in a 5 % CO2 atmosphere. Murine macrophages J774 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium GlutaMAX with 4.5 g/L d -glucose without sodium pyruvate at 37 °C in a 5 % CO2 atmosphere. Murine microglial BV-2 cells (American Type Culture Collection (ATCC), Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium GlutaMAX with 4.5 g/L d -glucose without sodium pyruvate at 37 °C in a 5 % CO2 atmosphere. Murine glioma SB28 cells and murine GL261 cells (DSMZ, German Collection of Microorganisms and Cell Cultures GmbH, Leibniz Institute, Braunschweig, Germany) were cultured in Dulbecco's modified Eagle's medium (DMEM) with l -glutamine, 4.5 g/L d -glucose, and 1 mM sodium pyruvate at 37 °C in a 10 % CO2 atmosphere. All the mediums were supplemented with 10 % fetal bovine serum (FBS) and 1.0 % antibiotics (penicillin/streptomycin).
6
Dendritic Cell-Based Immune Modulation
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DCs were generated from the bone marrow (BMDC) of 6 week-old C57BL/6 mice as described previously [17 ] using IL-4 and GM-CSF. Purified DCs at a concentration of 5 × 105 cells/ml were incubated with 100 μg of VCG or UV-EB (at an MOI of 10) in 24-well for 24 h. Culture supernatants were collected and assayed for cytokines (IL-12, IL-10, IL-4 and TNF-α) and harvested cells were stained for flow cytometric analysis.
JAWS II cells (ATCC, CRL-1194; Manassas, VA), an immortalized immature dendritic cell line, which was established from bone marrow cells of a p53-knockout C57BL/6 mouse [18 ] were used for VCG uptake experiments. Cells were cultured in complete culture medium consisting of IMDM with 10% FCS, 4 mM L-glutamine, 10 U/ml penicillin and 100 μg/ml streptomycin, 0.5 mM 2-ME, 1 mM sodium pyruvate, and 5 ng/ml murine GM-CSF essentially as described [19 (link)].
JAWS II cells (ATCC, CRL-1194; Manassas, VA), an immortalized immature dendritic cell line, which was established from bone marrow cells of a p53-knockout C57BL/6 mouse [18 ] were used for VCG uptake experiments. Cells were cultured in complete culture medium consisting of IMDM with 10% FCS, 4 mM L-glutamine, 10 U/ml penicillin and 100 μg/ml streptomycin, 0.5 mM 2-ME, 1 mM sodium pyruvate, and 5 ng/ml murine GM-CSF essentially as described [19 (link)].
7
Culturing Calu-3 and JAWSII Cell Lines
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Calu-3 cells (human lung adenocarcinomic bronchial epithelial cells) and JAWSII cells (mouse immature dendritic cells) were obtained from American Type Culture Collection (Manassas, VA, USA). Calu-3 cells were grown in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) and were cultured once a week; JAWSII cells were cultured in α-minimum essential medium supplemented with 20% FBS and 5 ng/ml recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF) and were subcultured once weekly. All the cell culture media contained 1% antibiotic/antimycotic liquid, and all cells were maintained at 37 °C in a 5% CO2 atmosphere.
8
CD40 Promoter Luciferase Assay
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The pGL3-luciferase reporter plasmid containing wild-type mouse CD40 promoter fragment (-505 to +22 region, represented here as p505-luc and previously described as D9 [8 (link)]) was provided by Prof. Herman Waldmann (University of Oxford, UK). The mutant CD40 promoter-luciferase constructs p505-MutR1-luc, p505-MutR2-luc and p505-Mut(R1+R2)-luc, in which the Runx-binding sites R1 (-489TGTGGT-484), R2 (-464TGCGGT-459) or both R1 and R2 were substituted with GAATTC sequence, were created by Mutagenex Inc. JAWSII cells (American Type Culture Collection; 5 x 105/well) were transfected with either of the above-mentioned reporter constructs (900 ng) together with a control renilla luciferase reporter pRL-CMV (100 ng) using the TransIT-2020 transfection reagent (Mirus). After 24 h, cells were either left untreated or treated for 24 h with LPS or TNFα. Luciferase activity was assayed with the dual-luciferase reporter assay kit (Promega).
9
Fluorescent Labeling and Transfection of Plasmid DNA
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Materials Plasmid EGFP-N1 (pEGFP; BD Clontech Laboratories, Inc., CA, U.S.A.), was amplified in Escherichia coli (DH5α) and purified using an Endfree Plasmid Maxi kit (Qiagen Sciences, MD, U.S.A.). The concentration of pEGFP was determined based on UV absorption at 260 nm. YOYO ® -1 Iodide (Life Technologies Japan Ltd., Tokyo, Japan) was used for fluorescent labeling of pEGFP. The STR-CH2SV40H2C peptides (Stearoyl-CHHPKKKRKVHHC) were synthesized and purified according to our previous paper. 9) JAWS II cells were purchased from American Type Culture Collection (WA, U.S.A.). Cell culture medium, minimum essential medium (MEM) alpha, certified fetal bovine serum (FBS), penicillin/streptomycin stock solutions, and 0.25% Trypsinethylenediaminetetraacetic acid (EDTA) were purchased from Life Technologies Japan Ltd.. Recombinant mouse granulocyte macrophage colony-stimulating factor (GM-CSF; Wako Pure Chemical Corporation, Osaka, Japan) was used to generate immature DCs.
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