MAF (HPLC: Purity ≥ 98%) was from Solarbio Life Sciences. The RAW264.7 cell line was purchased from Procell Life Science & Technology Co., Ltd. Dexamethasone (DEX) sodium phosphate injection was obtained from Huazhong Pharmaceutical Co., Ltd. The male SPF ICR mice were provided by Huabukang Biotechnology Co., Ltd. The product license number was SCXK 2019–0008, and the use license number was SYXK 2021–0005. The animal experiment was approved by the Ethnic Committee of Experiment Research Institute of Guizhou University of TCM. The TLR4 and myD88 primary antibodies were provided by Santa Cruz Biotechnology Inc. The NF‐κB and TNF‐α were from Proteintech Inc. The secondary anti‐mouse and anti‐rabbit antibodies were obtained from Beijing Zhongshan Golden Bridge. The mouse TNF‐α and IL‐6 ELISA kits were from Dakewe Biotech Co., Ltd.
Raw264.7 cell line
Manufactured by Procell
Sourced in China
The RAW264.7 cell line is a mouse monocyte/macrophage cell line derived from ascites of a tumor induced by the Abelson murine leukemia virus. This cell line is commonly used in biomedical research as a model for studying macrophage biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Clark Bioscience (1 mentions)
Dulbecco modified eagle medium (dmem), by Meilun (1 mentions)
Humidified incubator, by Heal Force (1 mentions)
Dulbecco modified eagle medium (dmem), by Procell (1 mentions)
Mouse msc medium, by Cyagen (1 mentions)
Puromycin, by Biosharp (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Solarbio (1 mentions)
Type 1 collagen, by BD (1 mentions)
Rpmi 1640 medium, by Solarbio (1 mentions)
Ifn γ, by BD (1 mentions)
12 protocols using raw264.7 cell line
1
Anti-inflammatory Effects of MAF
2
Culturing Macrophages and Schwann Cells
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Macrophages were cultured using the RAW264.7 cell line (CL‐0190, Procell Life Science & Technology, Wuhan, China) and Rma‐bm (R1920, Yaji Biological, Shanghai, China), starting from generation 4. Rat Schwann cells were cultured using the RSC96 cell line (CL‐0199, Procell Life Science & Technology, Wuhan, China) for passaging and tested starting from the 4th generation. The RAW264.7 cell and RSC cells medium consisted of 10% FBS (Clark Bioscience, Virginia, USA) and DMEM (Meilun, Dalian, China) and 1% triple antibodies (Meilun, Dalian, China) containing penicillin, streptomycin, and amphotericin. The Ram‐bm cell culture medium is a special culture medium for Ram‐bm, purchased from Yaji biological.
3
RAW 264.7 Cell Culture Protocol
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RAW 264.7 cell line was purchased from Procell Corp. (Wuhan, China) and cultured in DMEM (Procell, Wuhan, China). All the cultured cells were supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified incubator (Heal Force, Shanghai, China) with an atmosphere of 5% CO2 at 37 °C.
4
Cultivation of Mouse Cell Lines
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The mouse pre-osteoblast cell line MC3T3-E1 subclone 14, mouse ECs bEND.3, the Raw264.7 cell line and mouse BMSCs were obtained commercially from Procell Life Science & Technology (Wuhan, China). These cells were authenticated using the short tandem repeat method and proved negative for mycoplasma. Pre-osteoblasts were cultured in α-modified Eagle's medium (α-MEM) supplemented with 10% FBS, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. ECs were cultured in endothelial cell medium (ECM) (Cyagen Biosciences Inc., Guangzhou, China) prepared according to the manufacturer's protocol. Raw264.7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. Mouse MSCs were cultured in mouse MSC Medium (Cyagen) according to the protocol of the manufacturer. All cultured cells were maintained at 37 °C with 5% CO2. The medium was replaced every 3 days, and the cells were reseeded into suitable flasks when reaching 70–80% confluence.
5
Notch1 Knockdown in RAW264.7 Macrophages
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Mouse macrophages from the RAW264.7 cell line (Procell Life Science & Technology Co., Ltd., China) were cultured in low-glucose DMEM containing 10% FBS and 1% P/S in 5% CO2 at 37°C.
Lentivirus against Notch1 was obtained from Shanghai GeneChem Co., Ltd., China. Prior to transfection with lentivirus, RAW264.7 cells were seeded in 12-well plates at a 1 × 105 density per well, and fresh medium with lentivirus and HiTransG-P were added. After a 48-h transfection, 2 μg/ml puromycin (BS111, BioSharp, China) was added for the selection. After continuous selection with puromycin, the Notch1 knockdown RAW264.7 cell line was examined by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The lentivirus containing empty vector was used as a negative control. CON RAW264.7 cells and Notch1 knockdown RAW264.7 cells were stimulated with lipopolysaccharide (LPS) and cultured in DMEM with 5.5 or 50 mM glucose for 24 h. The cells were harvested for Western blotting and qRT-PCR analyses.
Lentivirus against Notch1 was obtained from Shanghai GeneChem Co., Ltd., China. Prior to transfection with lentivirus, RAW264.7 cells were seeded in 12-well plates at a 1 × 105 density per well, and fresh medium with lentivirus and HiTransG-P were added. After a 48-h transfection, 2 μg/ml puromycin (BS111, BioSharp, China) was added for the selection. After continuous selection with puromycin, the Notch1 knockdown RAW264.7 cell line was examined by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The lentivirus containing empty vector was used as a negative control. CON RAW264.7 cells and Notch1 knockdown RAW264.7 cells were stimulated with lipopolysaccharide (LPS) and cultured in DMEM with 5.5 or 50 mM glucose for 24 h. The cells were harvested for Western blotting and qRT-PCR analyses.
6
Eucommia ulmoides Polysaccharides Regulate RAW264.7 Cells
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Eucommia ulmoides polysaccharides (EUP, purity: 60%, Catalog No.S27810) was obtained from Shanghai Yeyuan biotech Co., Ltd., (Shanghai, China). The RAW 264.7 cell line was purchased from Procell Life Science Technology Co., Ltd. (Wuhan, China). RAW264.7 cells were seeded in 96-well plates with a cell concentration of 2 × 103 cells per well. The culture medium of macrophages is HyClone Dulbecco’s Modified Eagle Medium (DMEM, Gibco) + 5% fetal bovine serum (FBS, Gibco). The RAW264.7 cells were added to DMEM (control group) and DMEM containing different concentrations of EUP (10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml) and were cultured and recorded under a light microscope. On days 1, 3, and 5, cell proliferation experiments were performed with MTT. The absorbance was measured at 490 nm using a microplate reader (iMark, Bio-Rad).
7
RAW264.7 Cell Line LPS Stimulation
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RAW264.7 cell line was obtained from ProCell (Wuhan, China) and maintained in 37°C incubators with 5% CO2. The cultured media was DMEM media with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. RAW264.7 were pretreated with the indicated concentrations of CTS (2.5, 5, 10 μM) for 2 h before being stimulated with LPS (1 μg/mL) for another 24 h.
8
RAW264.7 Cell Line Cultivation
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Dulbecco’s Modified Eagle Medium (DMEM) was used to cultivate the RAW264.7 cell line, which was obtained from Procell Life Science & Technology in Wuhan, China. The medium was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, both of which were obtained from the same source. The RAW264.7 cells were cultured at 37 °C with 5% CO2 in a sterile, humidified cell incubator.
9
Culturing and Polarizing RAW 264.7 Macrophages
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RAW 264.7 cell line was purchased from Procell Life Science&Technology C, Ltd. (CL-0190, Procell, China). Cells were cultured in RAW 264.7 specialty medium (Procell, China) at 37 °C and 5% CO2. The medium contained 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (HyClone, Logan, USA). Consistent with previous literature reports, E. coli saccharide (LPS) and interferon-γ (IFN-γ) were used as M1 macrophage stimulating factors, and interleukin-4 (IL-4) as M2 macrophage stimulating factors [23 (link)].
10
Mouse Macrophage RAW264.7 Cell Culture
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The mouse macrophage RAW264.7 cell line was purchased from the Procell Life Science & Technology Co., Ltd. (Wuhan, China). According to the instructions, the cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Solarbio Science & Technology, Beijing, China) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Gaithersburg, MD, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Shanxi MiniBio Technology Co., Ltd, Shanxi, China) in a 5% CO2 incubator at 37°C. The medium was replaced the next day.
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