RAW 264.7 macrophage-like cells (ATCC, Manassas, VA, USA) were expanded in Dulbecco Modified Medium (DMEM) supplemented with 10% fetal bovine serum, antibiotics (all Invitrogen, Grand Island, NY, USA) and seeded 1 × 106 cells/cm2 into 96- and 24-well culture plates (VWR, Vienna, Austria). The next day, RAW 264.7 cells were exposed to 5% of ADL with or without TLR4 inhibitor, TAK-242 (Merck Millipore, Billerica, MA, USA) and lipopolysaccharide (LPS) from Escherichia coli 0111:B4 at 100 ng/mL (Sigma-Aldrich, St. Louis, MO, USA) for another 24 h under standard conditions at 37 °C, 5% CO2, and 95% humidity. Then, viability, RT-PCR and immunoassay were performed. For immunostainings, the protocol was adapted and cell exposure was one hour as stated below.
Raw264.7 macrophage like cells
Manufactured by American Type Culture Collection
Sourced in United States
RAW264.7 macrophage-like cells are a mouse monocyte/macrophage-like cell line derived from ascites of a tumor induced by the Abelson murine leukemia virus. They are commonly used as a model for studying macrophage biology and function.
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Lab products found in correlation
Tak 242, by Merck Group (1 mentions)
Lps from escherichia coli 0111 b4, by Merck Group (1 mentions)
Legendplex mouse inflammation panel, by BioLegend (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
M csf, by ProSpec (1 mentions)
Nih3t3 cells, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
B16bl6 cells, by Thermo Fisher Scientific (1 mentions)
Pcr mycoplasma detection set, by Takara Bio (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
K18 hace2 mice, by GemPharmatech (1 mentions)
6 protocols using raw264.7 macrophage like cells
1
Macrophage Activation and Inhibition Assay
2
Macrophage Inflammation Cytokine Assay
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RAW
264.7 macrophage-like cells were purchased from ATCC and cultured
according to instructions. One day after plating in a flat-bottom,
nontreated 96 well plate, cells were treated with various concentrations
of TLR7 equiv polymer or R848 (Sigma-Aldrich, cat. no. SML0196) (as
quantified by absorbance at 327 nm). Supernatant was collected 24
h after treatment and analyzed via LEGENDplex Mouse Inflammation Panel
(BioLegend, cat. no. 740446).
264.7 macrophage-like cells were purchased from ATCC and cultured
according to instructions. One day after plating in a flat-bottom,
nontreated 96 well plate, cells were treated with various concentrations
of TLR7 equiv polymer or R848 (Sigma-Aldrich, cat. no. SML0196) (as
quantified by absorbance at 327 nm). Supernatant was collected 24
h after treatment and analyzed via LEGENDplex Mouse Inflammation Panel
(BioLegend, cat. no. 740446).
3
Murine Osteoclast Differentiation Protocol
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Bone marrow cells were collected from the femora and tibiae of Balb/c mice aged 6–8 weeks old. Bone marrow cells were seeded at 3 × 106 cells/cm2 into 12-well plates and grown for 7 days in alpha Minimum Essential Medium (αMEM) supplemented with 10% fetal bovine serum, antibiotics (all Invitrogen, Grand Island, NY) and 30 ng/ml M-CSF (Prospec, Ness-Ziona, Israel). RAW264.7 macrophage-like cells (ATCC, Manassas, VA) were expanded in growth medium and seeded 1 × 106 cells/cm2 into 12-well plates under standard conditions at 37 °C, 5% CO2, and 95% humidity.
4
Circadian Rhythm Synchronization in Cell Lines
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RAW264.7 macrophage-like cells (RRID:CVCL_0493) were purchased from ATCC. B16/BL6 melanoma (RRID:CVCL_0157) and NIH3T3 fibroblasts (RRID:CVCL_0594) were purchased from Cell Resource Center for Biomedical Research (Tohoku University). RAW264.7 cells and NIH3T3 cells were cultured in DMEM supplemented with 5% FBS and 0.5% penicillin–streptomycin solution (Invitrogen; Life Technologies, Carlsbad, California). B16/BL6 cells were cultured in RPMI1640 supplemented with 5% FBS (Gibco BRL, Gaithersburg, Maryland) and 0.5% penicillin–streptomycin solution (Invitrogen). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. We confirmed the absence of microbes in these cell lines using a TaKaRa PCR Mycoplasma Detection Set. Cell lines were authenticated by each cell bank using short tandem repeat– PCR analysis, and these cell lines were used within 6 months from frozen stocks. To synchronize the cellular circadian clock, RAW264.7 cells were treated with 100 nmol/L dexamethasone (DEX) for 2 hours. Control cells were also set without treatment with DEX. The cells were washed with DMEM supplemented with 5% FBS and 0.5% penicillin and streptomycin, and then incubated in the medium at 37°C in a humidified 5% CO2 atmosphere. RNA and protein samples were prepared from circadian clock-synchronized cells every 4 hours after DEX treatment.
5
Generation and Culture of Murine Macrophages
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J774 cells and RAW 264.7 macrophage-like cells (American Type Culture Collection, Manassas, VA) were cultured and maintained in Dulbecco's Modified Eagle Medium supplemented with 10% (w/v) heat-inactivated fetal bovine serum, penicillin/streptomycin (50 units/ml) in a humidified atmosphere (5% CO2) at 37°C and used at low passage numbers. Cells were confirmed to be contamination-free. Bone marrow cells isolated from female C57BL/6 mice aged 6–13 weeks were differentiated for 7 days to generate BMMs by culture in the same media supplemented with 20% L-929-cell-conditioned media. Mice were housed in a pathogen-free environment at Weill Cornell Medical College and used in accordance with protocols approved by the Institutional Animal Care and Utilization Committees.
6
In Vitro and In Vivo Cell Models for Biomedical Research
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The murine cell lines of RAW 264.7 macrophage-like cells and DC2.4 DC-like cells were obtained from the American Type Culture Collection (ATCC) and cultured under the guidelines offered by the ATCC. The Institute of Cancer Research (ICR) mice (male, 6-week-old) and rattus norvegicus rats (female, 3-month-old) were both purchased from Hunan Silaike Jinda Laboratory Animal Co. Ltd. (China). The K18-hACE2 mice (14-week-old) were purchased from GemPharmatech Co. Ltd. (China). The rhesus macaques (male, 3- to 4-year-old) were purchased from Anhui Shengpeng Experimental Animal Technology Co. Ltd. (China). All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Shenzhen Bay Laboratory, Southern Medical University, and Chinese Academy of Agricultural Sciences in accordance with the guidelines for the protection of animal subjects.
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