Raw264.7 tib 71

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RAW264.7 (TIB-71) is a mouse macrophage cell line derived from the ascites of a tumor induced by the Abelson murine leukemia virus. It is commonly used in research as a model for studying macrophage biology and function.

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RAW264.7 (1261 citations) TIB-71 (120 citations) RAW264.7 cells (TIB-71) (9 citations) TIB-71TM (8 citations) RAW264.7 cells (ATCC TIB-71) (8 citations) RAW264.7 cells (ATCC number TIB-71) (4 citations) RAW264.7 murine macrophage cell line (TIB-71) (2 citations) RAW264.7 cell line (TIB-71) (2 citations)

36 protocols using raw264.7 tib 71

RAW264.7 Cell Culturing and Differentiation

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RAW264.7^TIB‐71 and RAW264.7^CRL‐2278 were purchased from ATCC. Cell properties are detailed in Table 1. The culturing and induction agents are detailed in Table 2. In our current study, RAW264.7^TIB‐71 cells were cultured as our previous studies reported.², ³ Briefly, RAW264.7^TIB‐71 was seeded into 96‐well plate by the density of 2 × 10³ cells/mL in DMEM, containing L‐glutamine (4 mM), FBS (10%), sodium bicarbonate (1.5 g/L) and glucose (4.5 g/L). Besides that, RAW264.7^CRL‐2278 was initially seeded into the 96‐well plate at the same density to RAW264.7^TIB‐71 (2 × 10³ cells/mL) in RPMI‐1640 (according to ATCC's instruction), which contains L‐glutamine (2 mM), FBS (10%), sodium bicarbonate (1.5 g/L) and glucose (4.5 g/L), HEPES (10 mM) and sodium pyruvate (1 mM) before the osteoclastic induction.

Kong L., Ma R., Cao Y., Smith W., Liu Y., Yang X, & Yan L. (2021). Cell cytoskeleton and proliferation study for the RANKL‐induced RAW264.7 differentiation. Journal of Cellular and Molecular Medicine, 25(10), 4649-4657.

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Monocytic cell culture protocol

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Mouse macrophage cell line RAW 264.7 (TIB-71™) and the human acute leukemia monocytic cells THP-1 (ATCC-88081201) were purchased from ATCC (American Type Culture Collection, Rockville, MD, USA). The cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum at 37 °C in a humidified 5% CO₂-containing atmosphere. THP-1 cells were differentiated (to dTHP-1) with 100 nM phorbol-12-myristate-13-acetate (PMA; Sigma Aldrich, Taufkirchen, Germany) for 72 h prior to each experiment and then the medium was exchanged with fresh medium without PMA. Passages between 10 and 30 were used for in vitro experiments.

Yildirim M., Weiss A.V, & Schneider M. (2023). The Effect of Elasticity of Gelatin Nanoparticles on the Interaction with Macrophages. Pharmaceutics, 15(1), 199.

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Murine and Human Pancreatic Cancer Cell Lines

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Murine pancreatic cancer cell line Pan02 (Panc02) was obtained from the Developmental Therapeutics Program at the NCI. The development of primary murine PDA cell lines (mPLR) is discussed in the supplement. Human pancreatic cancer cell lines (Capan-1 and MiaPaCa-2) were obtained from American Type Culture Collection (ATCC). Colo357 cells were obtained from Dr. Jason Fleming (MD Anderson Cancer Center). C5LM2 is a cell line derived from liver metastasis from a Panc-1 tumor bearing mouse isolated by our lab. RAW 264.7 (TIB-71) cells and NIH 3T3 cells were obtained from ATCC. Cell lines were confirmed to be pathogen-free and human cell lines were authenticated to confirm origin before use. Cells were cultured in DMEM (Invitrogen) or RPMI (Invitrogen) containing 10% fetal bovine serum and maintained at 37°C in a humidified incubator with 5% CO₂ and 95% air.

Ostapoff K.T., Cenik B.K., Wang M., Ye R., Xu X., Nugent D., Hagopian M.M., Topalovski M., Rivera L.B., Carroll K.D, & Brekken R.A. (2014). Neutralizing murine TGFβR2 promotes a differentiated tumor cell phenotype and inhibits pancreatic cancer metastasis. Cancer research, 74(18), 4996-5007.

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Murine Macrophage Cell Culture Protocol

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All chemicals and cell culture reagents were obtained from commercial sources and used without purification. Celecoxib was purchased from LC Laboratories, Woburn, MA, USA. Perfluoropolyethylene glycol ether (PFPE) was obtained as a generous gift from Celsense Inc., Pittsburgh, PA, USA. Pluronic® P105 was obtained from BASF, Cremophor EL® (CrEL) was obtained from Sigma Aldrich. 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine (DiD) dye was purchased from Life Technologies. Bacterial lipopolysaccharide (LPS) and cytochalasin B were obtained from Sigma Aldrich. The CellTiter-Glo® cell viability assay kit and Griess Reagent System for nitrite measurements were purchased from Promega, Madison, WI, USA. Phycoerythrin conjugated Rat anti-mouse CD86 (CD86-PE) antibody and Rat IgG_2a,_k conjugated to PE (isotype control) were obtained from BD Biosciences, San Jose, CA. The murine macrophage cell line RAW 264.7 (TIB-71) was obtained from ATCC. Cells were culture as previously reported [21 (link)]. Unless specified, cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

Patel S.K., Beaino W., Anderson C.J, & Janjic J.M. (2015). Theranostic Nanoemulsions for Macrophage COX-2 Inhibition in a Murine Inflammation Model. Clinical immunology (Orlando, Fla.), 160(1), 59-70.

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Evaluation of Swertiamarin's Anti-inflammatory and Anti-adipogenic Effects

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The RAW264.7 (TIB-71; ATCC, Manassas, VA, USA) murine monocytic cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% foetal bovine serum (FBS; Gibco) until the cells reached 90% confluence. Then, the cells were serum-starved for 6 h and incubated with 10 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) in the presence of swertiamarin for 16 h. The LPS-induced inflammatory signals were examined by western blotting.
3T3-L1 mouse pre-adipocytes (CL-173, ATCC) were cultured in DMEM supplemented with 10% FBS until confluent. At 2 days post-confluency, the medium was replaced with DMEM containing 0.5 mM methylisobutylxanthine (Sigma-Aldrich), 0.125 mM indomethacin (Sigma-Aldrich), and 1.0 µg/mL insulin (Sigma-Aldrich). After 2 days, the induced cells were cultured in maintenance medium (DMEM containing 10% FBS, 1.0 µg/mL insulin, and 1 nmol/L T3) for 5 days and treated with swertiamarin at the indicated concentrations for 48 h. Oil Red O staining and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the lipid content of cells.

Xu L., Li D., Zhu Y., Cai S., Liang X., Tang Y., Jin S, & Ding C. (2021). Swertiamarin supplementation prevents obesity-related chronic inflammation and insulin resistance in mice fed a high-fat diet. Adipocyte, 10(1), 160-173.

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Osteoclast Differentiation from RAW 264.7 and Bone Marrow Mononuclear Cells

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RAW 264.7 (TIB-71, mouse macrophage/monocytes) was purchased from ATCC. Cells were cultured in DMEM/1.5 g/L sodium bicarbonate (JRH Biosciences, Lenexa, KS) supplemented with 10% non-heat inactivated FBS (BioWhittaker, Cambrex, Walkersville, MD). To induce osteoclast differentiation, cells were cultured for 5 days in medium supplemented with 50 ng/ml of RANKL, (PeproTech Inc., Rocky Hill, NJ), with changes of medium and RANKL every other day. Bone marrow mononuclear cells (BMM) were collected from 2-week-old mice and cultured in a-MEM medium (Invitrogen, Carlsbad, CA) with 10% non-heat inactivated FBS (Bio Whittaker). To stimulate osteoclast differentiation, cells were cultured for 5 days in the presence of 50 ng/ml soluble RANKL (PeproTech Inc, Rocky Hill, NJ) and 25 ng/ml soluble M-CSF (PeproTech Inc.) with changes of medium, RANKL, and M-CSF every other day.

Battaglino R.A., Jha P., Sultana F., Liu W, & Morse L.R. (2019). FKBP12: a partner of Snx10 required for vesicular trafficking in osteoclasts. Journal of cellular biochemistry, 120(8), 13321-13329.

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Exosome Isolation from Mesenchymal Stromal Cells

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Primary human bone marrow mesenchymal stromal cells were purchased from ATCC (PCS-500–012). Cells were cultured in MSC culture media suggested by ATCC. P3-P6 cells were used for exosome isolation. Human primary coronary artery endothelial cells (HCAEC, PCS-100–020) and RAW 264.7 (TIB-71) were purchased from ATCC. Human cardiomyocytes (36044–15) were purchased from Celprogen Inc. Cell culture media were used as suggested.

Hu S., Wang X., Li Z., Zhu D., Cores J., Wang Z., Li J., Mei X., Cheng X., Su T, & Cheng K. (2021). Platelet membrane and stem cell exosome hybrid enhances cellular uptake and targeting to heart injury. Nano today, 39, 101210.

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Isolation and culture of human and mouse macrophages

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Monocytes were purified from buffy coat obtained from healthy blood donors, following a protocol approved by the Institutional Review Board at Shanghai Jiao Tong University. Briefly, human peripheral blood mononuclear cells (PBMCs) were separated by standard gradient centrifugation on Ficoll-Paque gradient. Monocytes were then isolated from PBMCs by positive sorting using anti-CD14-conjugated magnetic microbeads (Miltenyi, Germany). Monocyte purity was greater than 90% as assessed by flow cytometry (data not shown). Mouse bone marrow-derived macrophages (BMDMs) were prepared from bone marrow cells isolated from WT C57BL/6 mice or knockout mice as described previously (34 ). Human monocytic leukemia cell line THP-1 (TIB-202) and mouse macrophage cell line RAW264.7 (TIB-71) was purchased from ATCC (Manassas, VA). The cells were maintained in RPMI 1640 supplemented with 10% FBS, 1% streptomycin-penicillin and 10 μM β-mercaptoethanol. All cell cultures were kept at 37 °C in a humidified atmosphere with 5% CO₂.

Sun L., Zhou H., Zhu Z., Yan Q., Wang L., Liang Q, & Ye R.D. (2015). Ex vivo and in vitro effect of serum amyloid A in the induction of macrophage M2 markers and efferocytosis of apoptotic neutrophils. Journal of immunology (Baltimore, Md. : 1950), 194(10), 4891-4900.

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Characterization of P. gingivalis LPS

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Ultrapure P. gingivalis LPS (UP Pg LPS) (Invivogen, cat: tlrl-ppglps, lot: PPG-3701 and 3801), Standard P. gingivalis LPS (STD Pg LPS) (cat: tlrl-pglps, lot: LPG 37-01 and 38-01) were from InvivoGen (France). The STD Pg LPS were obtained by classical methods using hot-phenol extraction using the bacterial strain ATCC 33227 and UP Pg LPS preparations by enzymatic removal of lipoprotein from STD Pg LPS (Invivogen). Ultrapure lipopolysaccharide from E. coli O111:B4 (UP Ec LPS) (cat: tlrl-3pelps, lot: L3P 37-02) were from InvivoGen (France).
RAW264.7-Blue (RAW-Blue), HEK-Blue hTLR2-hCD14 (HEK-Blue hTLR2), HEK-Blue Null, HEK-Blue hTLR4-hCD14-hMD2 (HEK-Blue hTLR4), and HEK-Blue mTLR4-mCD14-mMD2 (HEK-Blue mTLR4) were from InvivoGen (France). BV2 was obtained as described previously^{35 (link)}. J774.1 (TIB-67), 3T3-L1 (CL-173), bEnd.3 (CRL-2299), and RAW264.7 (TIB-71) were from ATCC (USA), and all of these cells line express functional TLR2 and TLR4. TNF-α was from R&D systems (USA). Except when indicated, all other reagents were from Sigma-Aldrich (France). NFkB activity was performed using Quanti-blue assay following the manufacturer’s protocols (InvivoGen).

Nativel B., Couret D., Giraud P., Meilhac O., d’Hellencourt C.L., Viranaïcken W, & Da Silva C.R. (2017). Porphyromonas gingivalis lipopolysaccharides act exclusively through TLR4 with a resilience between mouse and human. Scientific Reports, 7, 15789.

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Culturing Human Kidney and Mouse Macrophage Cells

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Human embryonic kidney (HEK) 293 (#CRL-1572) and murine macrophage RAW 264.7 (#TIB-71) cells (ATCC, Manassas, VA) were grown in Dulbecco's Modification of Eagle's Medium (Mediatech, Inc, Manassas, VA) with 4.5 g/ L glucose, L-glutamine, sodium pyruvate, and supplemented with 10% heat inactivated fetal bovine serum (Mediatech Inc), 100 U/ mL penicillin (Mediatech Inc), and 0.1 mg/ mL streptomycin (Mediatech Inc).

Duellman T., Burnett J, & Yang J. (2015). Functional roles of N-linked glycosylation of human matrix metalloproteinase 9. Traffic (Copenhagen, Denmark), 16(10), 1108-1126.

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