Raw264 cells

Manufactured by RIKEN BioResource Center

Sourced in Japan

RAW264 cells are a mouse macrophage cell line derived from the BALB/c mouse strain. They are commonly used as a model for studying macrophage biology and function.

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Lab products found in correlation

Diff quik staining solution, by Sysmex (2 mentions) Isoflurane, by Fujifilm (2 mentions) Icr mice, by Charles River Laboratories (2 mentions) Luminal based chemiluminescent probe l 012, by Fujifilm (2 mentions) Fetal bovine serum (fbs), by Biowest (2 mentions) Raw 264, by Merck Group (1 mentions) Ethylene diamine tetraacetic acid (edta), by Nacalai Tesque (1 mentions) Lps from e coli o111 b4, by Merck Group (1 mentions) Rneasy kit, by Qiagen (1 mentions) Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions) Thunderbird sybr qpcr mix, by Toyobo (1 mentions) 3t3 l1 cells, by Health Science Research Resources Bank (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Ct26 cells, by RIKEN BioResource Center (1 mentions) Primescript rt master mix perfect real time, by Takara Bio (1 mentions) Thunderbird next sybr qpcr mix, by Toyobo (1 mentions) Fastgene rna basic kit, by Nippon Genetics (1 mentions) Ab21595, by Abcam (1 mentions) Ab5103, by Abcam (1 mentions)

Spelling variants of this product

RAW264.7 cells (26 citations) RAW264.7 cell line (8 citations) Murine RAW264.7 cells (2 citations) RAW264.7 mouse macrophage cells (2 citations)

11 protocols using raw264 cells

Culturing Raw264 Cells for Inflammation

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Raw264 cells (Riken BioResource Center) are cultured in 10 cm culture dishes and immersed in culture medium, and incubated as stated above. Cells are trypsinysed with a solution containing 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA, Nacalai) for approximately 5 min at 37 °C to detach them from the dish. They are then plated on quartz dishes at a density of 0.5 · 10⁵ cells/cm² and then incubated again as described above. Cells from passage number 12 to 18 were used for all experiments.
Then, 6–8 hours after plating, cell cultures (either primary cells or Raw264) are first rinsed with DMEM, which removes non-adherent cells in case of peritoneal cells, and cultures are then immersed in fresh medium containing LPS from E. Coli O111:B4 (Sigma-Aldrich) at 100 ng/mL.

Pavillon N, & Smith N.I. (2019). Immune cell type, cell activation, and single cell heterogeneity revealed by label-free optical methods. Scientific Reports, 9, 17054.

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Urban Aerosol-Induced Lung Injury Assessment

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Diff-Quik staining solution was purchased from Sysmex (Kobe, Japan). Antibodies against citrullinated histone H3 (citrulline R2 + R8 + R17) and neutrophil elastase were purchased from Abcam (Cambridge, UK). Luminal-based chemiluminescent probe (L-012) and isoflurane were obtained from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). The RNeasy® kit was obtained from Qiagen (Hilden, Germany), PrimeScript® 1st strand cDNA Synthesis kit was obtained from Takara Bio (Ohtsu, Japan), and THUNDERBIRD® SYBR qPCR Mix was obtained from Toyobo (Ohtsu, Japan). Novo-Heparin (5000 units), suitable for injection, was purchased from Mochida Pharmaceutical (Tokyo, Japan). RAW264 cells were purchased from Riken BioResource Center (Tsukuba, Japan). ICR mice (6–7 weeks old, male or female) were purchased from Charles River (Yokohama, Japan). The experiments and procedures described here were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and were approved by the Animal Care Committee of Musashino University. The sex of the mice did not correlate with the extent of urban aerosol-induced lung injury (number of inflammatory cells and levels of protein in bronchoalveolar lavage fluid (BALF)) (Supplementary Fig. S1). Therefore, male mice were used for all analyses in the present study.

Tanaka K.I., Kubota M., Shimoda M., Hayase T., Miyaguchi M., Kobayashi N., Ikeda M., Ishima Y, & Kawahara M. (2020). Thioredoxin-albumin fusion protein prevents urban aerosol-induced lung injury via suppressing oxidative stress-related neutrophil extracellular trap formation. Environmental Pollution (Barking, Essex : 1987), 268, 115787.

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Naringenin Modulates 3T3-L1 Adipocyte-Macrophage Crosstalk

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3T3-L1 cells (Health Science Research Resources Bank, Osaka, Japan) were maintained in DMEM supplemented were used for each group. Data are the mean ± SE. *P < 0.05 for STD vs HFD. § P < 0.05 for STD vs HFD + Nar. # P < 0.05 for HFD vs HFD + Nar. STD standard diet, HFD high-fat diet, Nar naringenin ◂ with 10% bovine serum. After pre-adipocytes reached confluence in 12-well plates, they were induced to differentiate into mature adipocytes by replacing medium with 10% FBSsupplemented DMEM containing 0.5 mM IBMX, 0.25 μM DEX, and 5 μg/mL insulin for 2 days. Medium was then replaced with 10% FBS-supplemented DMEM containing 5 μg/mL insulin, and this was changed every 2-3 days for the next 6-7 days. RAW264 cells (RIKEN BioResource Center, Tsukuba, Japan) were cultured in DMEM supplemented with 10% FBS. Adipocytes and macrophages were co-cultured in a contact system, as described previously (5) . Briefly, RAW264 cells (1 × 10 5 cells/mL) were plated onto dishes with differentiated 3T3-L1 adipocytes. For each experiment, we treated cells with naringenin and used 0.5% DMSO as the vehicle control. The total volumes of culture media used for the single cell line and the co-culture system treated with naringenin or vehicle control were 0.6 mL and 1.0 mL, respectively.

Tsuhako R., Yoshida H., Sugita C, & Kurokawa M. (2020). Naringenin suppresses neutrophil infiltration into adipose tissue in high-fat diet-induced obese mice. Journal of natural medicines, 74(1).

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Mouse Cell Lines Maintenance Protocol

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The RAW264 cells (a mouse leukemic monocyte cell line; ECA85062803) [11 (link)] were purchased from RIKEN BRC through the National BioResource Project of the MEXT, Japan. The RAW264 cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France), 100 U/ml penicillin, and 100 mg/ml streptomycin (Life Technologies, Carlsbad, CA, USA). The immortalized mouse myoblast cell line, C2C12 cells (ATCC CRL-1772) [12 (link)], was obtained from the JCRB (Japanese collection of research bioresources) cell bank (Tokyo, Japan). The C2C12 cells were maintained in D-MEM medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. These cells were grown at 37°C in 5% CO₂ in a humidified incubator.

Uchiyama H., Muramatsu D., Higashi H., Kida H, & Iwai A. (2023). Effects of chondroitin sulfate oligosaccharides on osteoclast differentiation of RAW264 cells, and myotube differentiation of C2C12 cells. PLOS ONE, 18(4), e0284343.

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Murine Macrophage Isolation and Treatment

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Macrophages were obtained from murine bone marrow cells according to published methods [14 ], with minor modifications. Briefly, bone marrow was harvested from the femur and tibia. Red blood cells were removed by hypotonic burst, and bone marrow cells were suspended in RPMI1640 medium supplemented with 10% fetal bovine serum albumin, 2 mM L-glutamine, 100 μg mL⁻¹ streptomycin, and 100 U mL⁻¹ penicillin. Cells were grown at 37°C, 5% CO₂, and 5×10⁵ cells mL⁻¹. Cultures were supplemented on day 0 with 20 ng mL⁻¹ recombinant murine macrophage colony stimulating factor. On day 3, half of the medium was replaced with fresh medium supplemented with the colony-stimulating factor. On day 5, adherent proliferating macrophages were harvested, purified, and cultured for 24 h at 5×10⁵ cells well⁻¹, with or without of 10 μM haloperidol. Finally, cells were stimulated with 100 ng mL⁻¹ LPS for another 24 h.
RAW 264 cells, which are macrophages isolated from Balb/c mice and immortalized with Abelson leukemia virus, were purchased from RIKEN BioResource Center (Tsukuba, Japan). Cells were cultured in complete media at 37°C, 5% CO₂ and 5×10⁴ cells well⁻¹. On day 2, cells were treated with 0.5, 5, or 10 μM haloperidol for 24 h, and then exposed to 300 ng mL⁻¹ LPS for another 24 h.

Yamamoto S., Ohta N., Matsumoto A., Horiguchi Y., Koide M, & Fujino Y. (2016). Haloperidol Suppresses NF-kappaB to Inhibit Lipopolysaccharide-Induced Pro-Inflammatory Response in RAW 264 Cells. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 367-372.

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Diosgenin Modulates Macrophage Inflammation

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Mouse macrophage-like RAW264 cells was obtained from RIKEN BioResource Center (Tsukuba, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. RAW264 cells were incubated with or without 100 nM diosgenin. After the incubation for 3 h, the cells were stimulated by 2.5 µg/ml lipopolysaccharide (LPS) for 6 h. mRNA expression of Ptgs2 and Ptges was measured by quantitative RT-PCR.

Tsukayama I., Toda K., Takeda Y., Mega T., Tanaka M., Kawakami Y., Takahashi Y., Kimoto M., Yamamoto K., Miki Y., Murakami M, & Suzuki-Yamamoto T. (2018). Preventive effect of Dioscorea japonica on squamous cell carcinoma of mouse skin involving down-regulation of prostaglandin E2 synthetic pathway. Journal of Clinical Biochemistry and Nutrition, 62(2), 139-147.

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Murine Macrophage and Colon Carcinoma Cell Cultures

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All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health (Bethesda, MD) and Guidelines for Animal Experiments of Ritsumeikan University (Shiga, Japan). The protocol was approved by the Animal Experimentation Committee of Ritsumeikan University (approval number: BKC2016-016). All efforts were made to minimize the suffering of experimental animals. Seven-week-old male Sprague Dawley rats were purchased from SLC, Inc. (Shizuoka, Japan).
RAW264 cells, a murine macrophage-like cell line, and CT-26 cells, a murine colon carcinoma cell line, were obtained from Riken Bioresource Center (Ibaraki, Japan). The cells were maintained in RPMI 1640 medium supplemented using 10% heat-inactivated fetal bovine serum (FBS), penicillin G (100 U/mL), and streptomycin (100 µg/mL) at 37 °C in 5% CO₂/95% air.

Kono Y., Nakai T., Taguchi H, & Fujita T. (2017). Development of magnetic anionic liposome/atelocollagen complexes for efficient magnetic drug targeting. Drug Delivery, 24(1), 1740-1749.

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Murine Macrophage Cell Culture

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RAW264 cells (a murine macrophage line) were obtained from the Cell Bank RIKEN BioResource Center (Tsukuba, Japan). Cells were grown in MEM supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cultures were maintained at 37°C in a 5% CO₂ humidified atmosphere.

Ohta T., Ido A., Kusano K., Miura C, & Miura T. (2014). A Novel Polysaccharide in Insects Activates the Innate Immune System in Mouse Macrophage RAW264 Cells. PLoS ONE, 9(12), e114823.

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Neutrophil Activation and Histone Citrullination

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Diff-Quik staining solution was purchased from Sysmex (Kobe, Japan). Antibodies against citrullinated histone H3 (citrulline R2 + R8 + R17) (ab5103) and neutrophil elastase (ab21595) were purchased from Abcam (Cambridge, UK). An antibody against myeloperoxidase (22225-1-AP) was purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Luminal-based chemiluminescent probe (L-012) and isoflurane were obtained from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). A FastGene™ RNA Basic kit was obtained from Nippon Genetics Co., Ltd. (Tokyo, Japan), PrimeScript™ RT master mix (Perfect Real Time) was obtained from Takara Bio (Shiga, Japan), and THUNDERBIRD^® Next SYBR^® qPCR mix was obtained from Toyobo (Osaka, Japan). Novo-Heparin (5000 units) suitable for injection was purchased from Mochida Pharmaceutical (Tokyo, Japan). RAW264 cells were purchased from Riken BioResource Center (Tsukuba, Japan). ICR mice (6–7 weeks old, male) were purchased from Charles River (Yokohama, Japan). The experiments and procedures described herein were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and were approved by the Animal Care Committee of Musashino University.

Tanaka K.I., Nakaguchi S., Shiota S., Nakada Y., Oyama K., Sakakibara O., Shimoda M., Sugimoto A., Ichitani M., Takihara T., Kinugasa H, & Kawahara M. (2022). Preventive Effect of Epigallocatechin Gallate, the Main Component of Green Tea, on Acute Lung Injury Caused by Air Pollutants. Biomolecules, 12(9), 1196.

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Culturing RAW264 Macrophage Cells

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RAW264 cells (Resource No. RCB0535) were purchased from RIKEN BioResource Center (RIKEN BRC, Tsukuba, Ibaraki, Japan). RAW264 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Merck KGaA, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Bio West, Nuaillé, France) and penicillin-streptomycin solution (Merck KGaA, Darmstadt, Germany) at 37 °C in a humidified incubator containing 5% CO₂ using 75 cm² flask (BD FALCON, Corning, NY, USA).

Takahashi S., Ferdousi F., Yamamoto S., Hirano A., Nukaga S., Nozaki H, & Isoda H. (2023). Botryococcus terribilis Ethanol Extract Exerts Anti-inflammatory Effects on Murine RAW264 Cells. International Journal of Molecular Sciences, 24(7), 6666.

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