To investigate the role of AT-EVs in inflammation, 5 × 105 RAW 264.7 macrophages were seeded in six-well plates and cultured with high-glucose DMEM with FBS, until cells reached 80% confluence. As previously described (Xu et al., 2020 (link)), cells were divided into five groups for which the culture medium was replaced as follows: Control group : cells were cultured with DMEM; Lipopolysaccharide (LPS) + IFNγ group : cells were cultured with DMEM +1 μg/mL LPS (#L2880, Sigma-Aldrich, St. Louis, MO, United States) + 30 ng/mL IFNγ (#315-05-100, PeproTech, Rocky Hill, NJ, United States); LPS + IFNγ + dAT-EVs group : cells were cultured with 1 μg/mL LPS +30 ng/mL IFNγ +150 μg/mL dAT-EVs; LPS + IFNγ + mAT-EVs group : cells were cultured with 1 μg/mL LPS +30 ng/mL IFNγ +150 μg/mL mAT-EVs; LPS + IFNγ + cAT-EVs group : cells were cultured with 1 μg/mL LPS +30 ng/mL IFNγ +150 μg/mL c AT-EVs. After induction for 24 h, 2 × 105 cells were incubated with fluorescein isothiocyanate (FITC)-anti-mouse CD86 (1:50, #105110, BioLegend, San Diego, CA, United States) and PE-anti-mouse CD206 (1:40, #141706, BioLegend) at 4°C for 30 min, before being washed twice with staining buffer (#00-4222-26, Invitrogen, San Diego, CA, United States of America). Polarization of RAW 264.7 cells was determined using flow cytometry (BD FACSCalibur, Beckman Coulter, Inc., Brea, CA, United States).
Ifn γ
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, China, France, Japan, Italy, Canada, Austria, Macao, Switzerland, Spain, Sao Tome and Principe, Israel, Sweden, Lao People's Democratic Republic
IFN-γ is a laboratory reagent used to detect and measure interferon-gamma, a cytokine protein that plays a key role in the immune response. It is often used in cell-based assays and immunoassays to quantify IFN-γ levels in biological samples.
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Lab products found in correlation
Interleukin 4 (il 4), by Thermo Fisher Scientific (392 mentions)
Tnf α, by Thermo Fisher Scientific (326 mentions)
Lipopolysaccharides (lps), by Merck Group (245 mentions)
Il 1β, by Thermo Fisher Scientific (139 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (138 mentions)
Il 10, by Thermo Fisher Scientific (138 mentions)
Il 13, by Thermo Fisher Scientific (136 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (96 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (85 mentions)
Il 17a, by Thermo Fisher Scientific (79 mentions)
M csf, by Thermo Fisher Scientific (78 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (71 mentions)
Gm csf, by Thermo Fisher Scientific (70 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (63 mentions)
Il 17, by Thermo Fisher Scientific (57 mentions)
Tgf β, by Thermo Fisher Scientific (53 mentions)
Streptomycin, by Thermo Fisher Scientific (52 mentions)
Penicillin, by Thermo Fisher Scientific (51 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (40 mentions)
Phorbol myristate acetate (pma), by Merck Group (39 mentions)
1 815 protocols using ifn γ
1
Investigating AT-EV Modulation of Macrophage Polarization
2
Cytokine Stimulation of Splenocytes
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Spleens were aseptically removed from naïve WT, Il21r-/-, STAT3flox/flox or CD4stat3-/- mice and cells were mechanically disrupted through a 100 μm cell strainer (BD Biosciences, San Jose, CA) using the plunger of a 6 ml syringe. RBCs were lysed in ACK lysing buffer (Lonza, Walkersville, MD), and the remaining cells were washed twice with ice-cold PBS. A total of 5×106 splenocytes were cultured in duplicate in 1 ml of RPMI 1640 (Gibco) supplemented with 10% FBS and 100 μg/ml penicillin/streptomycin (all from Gibco). Cells were allowed to rest for 2 additional hours at 37°C and subsequently were stimulated in the presence of recombinant murine IFN-γ, IL-17A, IL-21 and IL-22 (20 ng/ml; Peprotech, Rocky Hill, New Jersey, USA) alone or in combination (IFN-γ+IL-17A; IFN-γ+IL-21; IFN-γ+IL-22; 20 ng/ml each). Cells receiving no cytokine treatments were used as controls. Cells were harvested at different intervals following the addition of cytokines and were stained with indicated antibodies.
3
ADSC-EVs Regulate Macrophage Polarization
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To investigate the role of ADSC-EVs in regulating macrophage polarization in an inflammatory environment, 5 × 105 raw 264.7 cells were seeded in 6-well plates. After cell attachment, the culture medium was replaced with culture medium (control group), culture medium containing 1 mg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng/mL IFN-γ (PeproTech, Rocky Hill, USA) (LPS + IFN-γ group), or culture medium containing 1 mg/mL LPS, 20 ng/mL IFN-γ, and different concentrations of EVs (LPS + IFN-γ + EV group), as previously described [23 (link)]. After incubating the different media for 24 h, the polarization of raw 264.7 cells to M1/M2 macrophages was identified by flow cytometry, immunofluorescence staining, and quantitative real-time polymerase chain reaction (qPCR).
4
Investigating CEFFE's Role in Macrophage Polarization
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To investigate the role of CEFFE in regulating macrophage polarization in an inflammatory environment, 4 × 105 Raw 264.7 cells were seeded in 6-well plates. After 24 h of cell contact, the normal medium was replaced with 2 mL culture medium (control group); culture medium containing 1 µg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, USA) and 30 ng/mL interferon-γ (IFN-γ; PeproTech, Rocky Hill, USA) (LPS + IFN-γ group); culture medium containing 1 µg/mL LPS, 30 ng/mL IFN-γ; and different concentrations of CEFFE (100 µg/mL, 250 µg/mL, and 500 µg/mL). After incubating in these media for 24 h, all media were replaced with fresh media for another 24 h. The polarization of Raw 264.7 cells to M1/M2 macrophages was identified by flow cytometry, immunofluorescence staining, and quantitative real-time polymerase chain reaction (qRT-PCR). We also tested the effects of different concentrations of CEFFE on inactive macrophages in the same way.
5
Genome-wide CRISPR screen for IFNγ resistance
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A custom gRNA library was manually designed from an extensive literature search, generated (Oxford Genetics), titrated using an mCherry fluorophore, and used at a viral titer that achieved 30–50% infection in HT-29 and LS-411N cells stably expressing Cas9.34 (link) Cells were selected with puromycin for 4 d (2 μg/mL and 1 μg/mL, respectively), maintaining 300 X coverage, with a time 0 (T0) control sample taken 7 d after infection. 10 d after infection, cells were selected with IFNγ (2000 U/mL; Thermo Fisher Scientific) for a total of 7 d with the IFNγ arm having IFNγ media refreshed after 4 d and the control arm being passaged after 4 d. Each screen was performed independently twice on separate days.
6
Cytokine Quantification in Muscle Extracts
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96-well microplates were coated by 0.2 μg antibodies against IL-1α (R&D systems), IL-13 (R&D systems), IFN-γ (eBioscience) and TNF-α (eBioscience), respectively. Total proteins extracted from TA muscles were applied to the microplates. Biotinylated goat antibodies against IL-1α (R&D systems), IL-13 (R&D systems), IFN-γ (eBioscience) and TNF-α (eBioscience) were applied as detection antibodies, respectively. Streptavidin-HRP antibody (R&D systems) was used as the secondary antibody. The signals were collected by microplate reader (Thermo MK3, Thermo Fisher) and quantified by comparison with standard solution containing recombinant mouse IL-1α (R&D systems), IL-13 (R&D systems), IFN-γ (eBioscience) and TNF-α (eBioscience), respectively. The standard curves were made according to the manufacturer's instructions.
7
Clonal Selection and IFN-γ Stimulation
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Cells were transfected using jetPEI (Polyplus-transfection SA) according to the manufacturer’s protocol in a 24-well cell plate (TPP), and maintained in medium with 100 μg/ml blasticidin (InvivoGen). Cloning was performed by adding ~200 cells to a 150 mm cell culture dish (TPP) to ensure separation of single cells. After 3 weeks, colonies were transferred to a 6-well cell plate (TPP) using cloning filters (Sigma-Aldrich). A share of each of the clones was induced for 72 h with 25 ng/ml IFN-γ (Life Technologies) in a 6-well plate (TPP) and analyzed by direct flow cytometry (see below). Clones were considered positive for the transfected construct if the mean fluorescence intensity exceeded that of the isotype control by 50%, and positive clones were divided into 2 separate wells in a 6-well cell plate (TPP), and either stimulated with 25 ng/ml IFN-γ (Life Technologies) for 72 h or left un-induced, before quantitative flow cytometry (see below).
8
Cytokine Quantification by Sandwich ELISA
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The concentrations of IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ in supernatants were quantified by sandwich ELISA using the following anticytokine antibody pairs: IL-4 (Biosource; AHC0642 and AHC0749), IL-5 (BioLegend; 500902 and 501002), IL-10 (eBioscience; 14-7108-85 and 13-7109-85), IL-13 (Invitrogen; P130E and M130B), IL-17 (eBioscience; 14-7178-85 and 13-7179-85), and IFN-γ (eBioscience; 14-7318-85 and 13-7319-85). The capture antibody was coated in NUNC MaxiSorp plates (Greiner Bio-One) overnight at 4°C in 0.05 mol Na2CO3 buffer (pH 9.7) (Merck, Boston, Mass). The plates were blocked with 1% BSA for 1 hour at 37°C. Supernatants and biotinylated antibodies were added and plates were incubated for 2 hours at room temperature.
9
Differentiation and Activation of Primary Macrophages
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Primary macrophages were derived as previously described (3 (link)). Briefly, marrow was flushed from the femurs and tibias of 2- to 4-month-old 129SvEvTac mice (Taconic Laboratories, Hudson, NY). Cells were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) supplemented with fetal bovine serum (10%), l -glutamine (2 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 µM), HEPES (10 mM), and penicillin-streptomycin (50 IU/ml penicillin and 50 µg/ml streptomycin). Cells were overlaid onto an equal volume of Histopaque-1083 (Sigma-Aldrich, St. Louis, MO) and centrifuged at 500 × g for 15 min. Monocytes at the interface were harvested and incubated for 6 days at 37°C in 5% CO2 in supplemented DMEM that also contained 30% macrophage colony-stimulating factor (M-CSF) (conditioned medium from 3T3-MCSF cells) to promote differentiation of adherent monocytes into macrophages (54 (link)). BMDM and RAW264.7 Nramp1G169 cells were activated with 20 ng/ml S. enterica serovar Typhimurium LPS from Sigma-Aldrich (where the dose is equivalent to a multiplicity of infection [MOI] of ~5,000 [20 pM] based on an E. coli dry weight of 2.8e−13 g/cell [55 ], 4.09 nmol LPS/mg dry weight of S. Typhimurium [56 (link)], and the formula weight of S. Typhimurium LPS [Sigma-Aldrich]) and 20 U/ml IFN-γ (PeproTech, Rocky Hill, NJ) or with only 20 U/ml IFN-γ for 18 h.
10
Macrophage differentiation and stimulation
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Raw264.7 cells were cultured in RMPI-1640 containing 20% (vol/vol) FBS (GIBCO, Invitrogen Inc, Carlsbad, CA, USA) and 1% (vol/vol) antibiotics (100 U/ml penicillin) at 37 °C in 5% CO2. Raw264.7 stimulated with IL-4 (25 ng/ml; catalog no. 214-14; PeproTech, Rocky Hill, NJ, USA), or with IFN-γ (25 ng/ml; catalog no. 315-05; PeproTech, Rocky Hill, NJ, USA) and LPS (100 ng/ml; catalog no. L2630; Sigma, St Louis, MO, USA) for 24 h. Adherent cells were washed and harvested with trypsin/EDTA (Lonza).
Bone marrow-derived macrophages (BMMs) were isolated, as previously described [60 (link)]. BMMs obtained from Lyz2-Cre + Twist1fl/fl and Lyz2-Cre-Twist1fl/fl mice were cultured in RMPI-1640 containing 10% (vol/vol) FBS, 25 ng/ml mouse M-CSF (catalog no. 315-02; PeproTech, Rocky Hill, NJ, USA), and 1% (vol/vol) penicillin/streptomycin antibiotics for 5 days, Briefly, on day 5, cells were replated in triplicate (3 × 105 cells/well). BMMs were cultured with serum-free medium and treated with IL-4 (25 ng/ml; catalog no. 214-14; PeproTech, Rocky Hill, NJ, USA), or with IFN-γ (25 ng/ml; catalog no. 315-05; PeproTech, Rocky Hill, NJ, USA) and LPS (100 ng/ml; catalog no. L2630; Sigma, St Louis, MO, USA) for 24 h. Adherent cells were washed and harvested with trypsin/EDTA (Lonza).
Bone marrow-derived macrophages (BMMs) were isolated, as previously described [60 (link)]. BMMs obtained from Lyz2-Cre + Twist1fl/fl and Lyz2-Cre-Twist1fl/fl mice were cultured in RMPI-1640 containing 10% (vol/vol) FBS, 25 ng/ml mouse M-CSF (catalog no. 315-02; PeproTech, Rocky Hill, NJ, USA), and 1% (vol/vol) penicillin/streptomycin antibiotics for 5 days, Briefly, on day 5, cells were replated in triplicate (3 × 105 cells/well). BMMs were cultured with serum-free medium and treated with IL-4 (25 ng/ml; catalog no. 214-14; PeproTech, Rocky Hill, NJ, USA), or with IFN-γ (25 ng/ml; catalog no. 315-05; PeproTech, Rocky Hill, NJ, USA) and LPS (100 ng/ml; catalog no. L2630; Sigma, St Louis, MO, USA) for 24 h. Adherent cells were washed and harvested with trypsin/EDTA (Lonza).
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