All materials were purchased from vendors, including DPBS, DMEM (containing 4.5 g/L glucose, L-glutamine, and sodium pyruvate), FBS, and 0.25% trypsin containing 2.21 mM EDTA (Corning Cellgro, Manassas, VA, USA); LPS from E. coli 0111: B4 and BMH-21 (Sigma Aldrich, St. Louis, MO, USA); and penicillin (10,000 U/mL)/streptomycin (10,000 µg/mL) (Lonza, Walkersville, MD, USA). Mouse RAW264.7 macrophages were obtained from ATCC, ImKCs (immortalized Kupffer cells) were obtained from EMD Millipore Corporation (Temecula, CA, USA), and E. coli SOS-Chromotest kit was obtained from EBPI (Mississauga, Ontario, Canada).
Mouse raw264.7 macrophages
Manufactured by American Type Culture Collection
Sourced in United States
Mouse RAW264.7 macrophages are a well-established immortalized cell line derived from Mus musculus (mouse) macrophages. These cells are frequently used in research to study various aspects of macrophage biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Corning (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Corning (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
L929 cell, by American Type Culture Collection (1 mentions)
Jurkat t cells, by American Type Culture Collection (1 mentions)
J774a 1 macrophages, by American Type Culture Collection (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Red cell lysis buffer, by Merck Group (1 mentions)
Macrophage colony stimulating factor, by BioLegend (1 mentions)
Ifn γ, by BioLegend (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
5 protocols using mouse raw264.7 macrophages
1
Macrophage and Kupffer Cell Activation
2
Cell Culture and Plasmid Transfection Protocol
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Human embryonic kidney 293 T (HEK293T) cells, Jurkat T cells, mouse RAW264.7 macrophages, J774A.1 macrophages, and L929 cells were purchased from ATCC (Manassas, VA). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI 1640 (for Jurkat cells) supplemented with 10% heat-inactivated fetal bovine serum (FBS). All cell lines were verified to be mycoplasma-free. HEK293T cells were transfected with Lipofectamine 2000 reagent (Invitrogen) as described previously [12 (link)]. Plasmids expressing Flag-NLRP3, hemagglutinin (HA)-NEK7 (never in mitosis gene a-related kinase 7), Flag-ASC, Flag-procaspase 1 and Flag-pro-IL-1β were described previously [13 (link)]. pEGFP-parkin was from Prof. L. Wang (Wuhan University, Wuhan, China) [14 (link)].
3
Macrophage Polarization and Nanoparticle Uptake
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Mouse RAW 264.7 macrophages (ATCC, Manassas, VA, USA) were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 50 U/mL penicillin/streptomycin. Cells were grown at 37 °C and 5% CO2 in a water-jacketed incubator. For macrophage polarization and nanoparticle uptake experiment, cells were seeded in six-well plates at a density of 2 × 104 cell/cm2 supplemented with low FBS (1%). At 16 h after seeding, cells were treated with or without DGNS-GW or DGNS-Man (100 ng/mL) and with TNF-α (5 ng/mL, Life Technologies, Carlsbad, CA, USA) for three days with the daily renewal of culture media. After three days, cells were harvested with a 1 mL of TRIZOL reagent (Gibco-Invitrogen, Paisley, UK) for RNA isolation. Nanoparticle uptake post-TNF-α stimulation and DGNS-GW treatment was determined using black aggregate quantification. Black aggregates of DGNS-GW were visualized at high magnification to establish the number of cells incorporating the nanostars. The percentage of cells incorporating DGNS-GW was calculated as follows: the number of cells with black aggregates/total number of cells per field × 100. At least 30 different fields were used to calculate the uptake percentage per condition.
4
Bone Marrow-Derived Macrophage Isolation and Adoptive Transfer
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Bone marrow-derived macrophages (BMMCs) were generated as previously described.24 (link) Briefly, mononuclear cells were flushed from the femurs and tibias of C57BL/6 mice and cultured overnight. The non-adherent cells were collected and centrifuged at 1200 r.p.m. for 10 min. The cells were then treated with Red Cell Lysis Buffer (Sigma-Aldrich, St Louis, MO, USA). Mature macrophages were obtained from the differentiated mononuclear cells and stimulated with 20 ng/ml macrophage colony-stimulating factor (Biolegend, #576406) for 7 days. Mouse RAW264.7 macrophages were from the American Type Culture Collection (ATCC, Manassas, VA, USA). BMMCs and Mouse RAW264.7 macrophages were cultured in dulbecco's modified eagle medium with 10% fetal bovine serum (Gibco by Life Technologies, Bleiswijk, the Netherlands) and 1% penicillin/streptomycin. Cells were cultured at 37 °C with 5% CO2. For adoptive transfer, BMMCs stimulated with or without IL-25 (50 ng/ml, Biolegend, #587306), IL-15 (25 ng/ml, Biolegend) and interferon gamma (IFNγ; 50 ng/ml, Biolegend) for 2 days were collected in cold PBS, and 1 × 106 cells were adoptively transferred into every mouse intraperitoneally after a HFD for 3 months. The adoptive transfer was performed two times interspaced for a week, and all mice were killed for analysis 7 days after the second adoptive transfer experiment.
5
Gossypol Modulation of RAW264.7 Macrophage Gene Expression
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Mouse RAW264.7 macrophages were from American Type Culture Collection (Manassas, VA, USA). Cell culture reagents were from Gibco BRL (Thermo Fisher, Waltham, MA, USA). Gossypol (G8761-100MG, (+/−)-gossypol from cotton seeds, ≥95% pure) and dimethylsulfoxide (DMSO) were from Sigma (St. Louis, MO, USA). TRIzol was from Thermo Fisher (Waltham, MA, USA). SuperScript II reverse transcriptase, oligo(dT)12–18 primer, random primers, dNTPs, DTT and RNaseOUT were from Life Technologies. CFX96 real-time system-C1000 Thermal Cycler, 1× iQ SYBR Green Supermix and qPCR assay accessories were from Bio-Rad (Hercules, CA, USA). PCR primers were designed using Primer Express software (Thermo Fisher) and synthesized by Biosearch Technologies (Petaluma, CA, USA) (Supplementary Table S1 ).
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