RAW 264.7 cell line was purchased from ATCC. Murine bone marrow macrophages (BMM) were collected from the tibia of normal mice. BMMs and RAW cells were cultured in a -MEM/10% FBS with 1% gentamycin. RAW cells were grown on 60.8 cm2 plates, and passed when cells reached 75% confluence. RAW cells were kept under 30 passages for the experiment. BMMs were cultured and used once. For osteoclast differentiation, cells were seeded at a density of 1 x 10^4 cells/ well in 6-well plates. RAW cells were differentiated into osteoclasts in the presence of RANKL (100 ng/ml) for 4-5 days to generate osteoclasts. Osteoclast formation with BMMs were accomplished with RANKL (100 ng/ml) and M-CSF (50 ng/ml) addition to the culture medium for 4-5 days. Cells were fixed with acetone and stained for tartrate resistant acid phosphatase TRAP per manufacturer’s instructions. TRAP positive cells containing more than three or more nuclei were counted as osteoclasts.
Raw264.7 cell line
Manufactured by American Type Culture Collection
Sourced in United States, China, Poland, United Kingdom
The RAW264.7 cell line is a macrophage-like cell line derived from the ascites of a tumor induced in a male BALB/c mouse by the Abelson murine leukemia virus. This cell line is commonly used in immunology and cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (34 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (23 mentions)
Penicillin, by Thermo Fisher Scientific (14 mentions)
Streptomycin, by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Lipopolysaccharides (lps), by Merck Group (6 mentions)
Penicillin, by Merck Group (6 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (6 mentions)
Axio observer a1, by Zeiss (6 mentions)
Streptomycin, by Merck Group (5 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (4 mentions)
Hek293 cell line, by American Type Culture Collection (4 mentions)
Fetal bovine serum (fbs), by Euroclone (4 mentions)
Thp 1 cell line, by American Type Culture Collection (4 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharide lps, by Merck Group (3 mentions)
α mem, by Thermo Fisher Scientific (3 mentions)
Rankl, by Thermo Fisher Scientific (3 mentions)
201 protocols using raw264.7 cell line
1
Osteoclast Differentiation from RAW 264.7 and Bone Marrow Macrophages
2
Formulation Development and Characterization
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Pluronic® P105 was obtained from BASF Corporation. Cremophor EL (CrEL) was purchased from Sigma Aldrich. Miglyol 812N, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and the Raw 264.7 cell line were purchased from ATCC. Prostaglandin E2 EIA Kit-monoclonal was purchased from Cayman Chemical Company. DiR dye was from Life Technologies. Perfluoro(polyethylene glycol dimethyl ether) or PFPE oxide (CF3O(CF2CF2O)nCF3, where n=8–13) was obtained from Exfluor Research Corporation.
3
Culturing Diverse Cell Lines
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Mouse aortic endothelial cells (MAECs) were purchased from CHI Scientific, Inc. Raw 264.7 cell line was obtained from ATCC (American Type Culture Collection). Human fibroblast-like synoviocyte RA (HFLS-RA) cell line was purchased from Guangzhou Jennio-bio Co., Ltd. (Guangzhou, China). MAECs, HFLS-RA cells, and raw 264.7 cells were grown in Dulbecco modified eagle medium (DMEM) medium containing 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a humidified atmosphere with 5% CO2.
4
Murine RAW 264.7 Macrophage Activation
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The murine BALB/c derived RAW 264.7 cell line was obtained from ATCC (Manassas, Va). The cells maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) which was supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% Penicillin-Streptomycin (15-140-122, Gibco). This base media formulation serves as the M0 control media. Cells were routinely subcultured every 3–4 days and maintained at 37 °C in 5% CO2. Macrophage activations was assessed by seeding at 1.05 × 104 cells/cm2 and allowing them to recover overnight. M1 stimulation media was M0 control media with 10 ng/ml IFNγ (Peprotech) and 100 ng/ml Lipopolysaccharide (LPS) (SigmaAldrich) added. M2 stimulation media was M0 media with 40 ng/ml IL-4 (Peprotech) and 20 ng/ml IL-13 (Peprotech) added. Stimulation with M1 media, M2 media, and silk particles occurred 24 hours after seeding. 250 μl of stimulation media was added to each well with unstimulated wells receiving M0 media. The wells stimulated with silk nanoparticles received 250 μl of M0 media supplemented with 50 μg of silk particles. 48 hours post-stimulation the media was changed using the same conditions as the first stimulation. This stimulation strategy resulted in 15 groups 72 hours post-stimulation: M0 (unstimulated), M1-Like, M2-Like, and the 12 silk nanoparticle formulations.
5
RAW 264.7 Cell Viability Assay
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A RAW 264.7 cell line (ATCC, Manassas, VA) was grown in RPMI- 1640 medium (Corning, USA) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM stable L-glutamine. RAW 264.7 cells were maintained under standard conditions in a humidified atmosphere of 5% CO2 at 37 °C. The cells were grown in a 96-well plate at a density of 3.0 × 105 cell/cm2 for 24 h. Cells were treated with the filter-HA, hotplate-HA and microwave-HA after calcination at 600 °C and commercial at various concentrations (0–500 μg/mL) for 24 h. After that, the spent medium was then removed and 200 μl of 0.5 mg/ml 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO) solution was added into each well and incubated at 37 °C in a CO2 incubator for 3 h. The resulting formazan crystals were dissolved in DMSO, and the absorbance for each well was detected at 560 nm using a microplate reader (Thermo Fisher, USA). Data were obtained from three independent experiments in four replicates. Cell viability was calculated using the following equation:
6
Measuring Transcription Factor Activity in RAW264.7 Cells
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The RAW264.7 cell line (ATCC, Manassas, Virginia, USA) was stably transfected with NF-κB 35 (link) and NFATc1 luciferase reporter gene constructs 36 (link), and transiently with Nrf2-ARE luciferase reporter gene construct, and then seeded in 48-well plates at the concentration of 1.5×105, 5×104 and 1×105 cells per well respectively. Cells were cultured overnight and then pre-treated with LrB for 1 hour and stimulated with RANKL for 6, 24 and 12 hours respectively. After stimulation, cells were lysed using luciferase lysis buffer and luciferase activities were measured using a luciferase reporter assay kit (Promega, Sydney, NSW, Australia, Cat# 318248) and a luminescence plate reader (BMG LABTECH, Ortenberg, Germany).
7
Investigating Macrophage Polarization in IDD
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Human NP cells were isolated from IVD of patients with IDD using the method previously described by Risbud et al. The isolated NP cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum supplemented with antibiotics. To investigate macrophage polarization, the conditioned medium (CM) of human primary NP cells from patients with IDD were collected. RAW264.7 cell line was purchased from ATCC.
8
Investigating miR-93 in RAW 264.7 Macrophages
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RAW 264.7 macrophages are widely used as a model of LPS-induced ALI, and play an essential role in the regulation of the inflammatory response (30 (link),31 (link)). Therefore, RAW 264.7 cells were selected for use in an ex vivo experiment as the present study focused on the ALI-induced inflammatory response. The RAW264.7 cell line was obtained from ATCC. The cells were maintained in DMEM supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA), 1% penicillin and streptomycin (Sigma-Aldrich; Merck KGaA) at 37°C and a 5% CO2 incubator.
When the RAW264.7 cells in a 6-well plate grown to approximately 80% confluency, miR-93 mimics (20 nmol/l) and miR-93 inhibitor (20 nmol/l) were transfected into the cells at 37°C for 24 h, using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). miR-93 mimics, miR-93 inhibitor and the corresponding control vectors were purchased from RiboBio Co., Ltd.
When the RAW264.7 cells in a 6-well plate grown to approximately 80% confluency, miR-93 mimics (20 nmol/l) and miR-93 inhibitor (20 nmol/l) were transfected into the cells at 37°C for 24 h, using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). miR-93 mimics, miR-93 inhibitor and the corresponding control vectors were purchased from RiboBio Co., Ltd.
9
Macrophage Modulation by bm-MSC CM
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The RAW264.7 cell line was purchased from ATCC and cultured under conventional culture conditions. The purity of cells was detected by flow cytometry using F4/80 labeling. After 24 h of adherent culturing, the macrophage culture medium RPMI-1640 was replaced with the CM of bm-MSCs in each group (the preparation of CM is described in the previous ‘Cell culture’ section), and then continued to be cultured for another 3 days.
10
Formulation and Evaluation of Praziquantel Nanoparticles
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Glyceryl behenate (Compritol 888 ATO) was procured as a free sample from Gattefosse SAS (Saint-Priest, CEDEX, France). Praziquantel was purchased from LeapChem Co. Ltd.(Hong Kong, China), L-α-lecithin soybean which is composed of less than 2% triglycerides and 94% phosphatidylcholine). PF127, phosphate-buffered, 49409 Atto 488 Phalloidin, was bought from Sigma-Aldrich (St. Louis, MO, USA). RAW 264.7 cell line was procured from ATCC (Manassas, VA, USA), and ethanol and chloroform were obtained from Merck Pty Ltd. (Modderfontein, South Africa). The DAPI stain were purchased from Thermofisher (Waltham, MA, USA). All other components and chemicals utilized in this study were of analytical grade. For all the preparations in the study, double deionized water (DDW) was employed. Biochemical assays (AST, ALT, ALP, bilirubin, and creatinine) kits were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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