Preparation of M1- and M2a-like macrophages was performed according to previously established methods58 (link). Briefly, bone marrow cells were harvested from the femur and tibia of AIM+/+ and AIM−/− mice, injected into ice-cold RPMI 1640 medium (Sigma-Aldrich), and filtered through a 70-μm nylon mesh. One μl of LPS (Sigma-Aldrich, 4 mg/ml) and 1.5 μl of IFN-γ (20 ng/ml; Cell Signaling Technology, Inc., Danvers, MA) or 2 μl of IL-4 (20 ng/ml; Cell Signaling Technology, Inc.) and IL-13 (20 ng/ml; Cell Signaling Technology, Inc.) were used to transform the phenotype to M1 or M2a character, respectively.
Interleukin 4 (il 4)
Manufactured by Cell Signaling Technology
Sourced in United States
IL-4 is a recombinant protein produced by Cell Signaling Technology. It is a cytokine that plays a key role in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Il 1β, by Cell Signaling Technology (3 mentions)
Ifn γ, by Cell Signaling Technology (2 mentions)
Il 13, by Cell Signaling Technology (2 mentions)
Il 10, by Cell Signaling Technology (2 mentions)
Nf κb, by Cell Signaling Technology (2 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Interleukin 4 (il 4), by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
Gm csf, by Cell Signaling Technology (1 mentions)
Elisa brdu colorimetric immunoassay, by Roche (1 mentions)
Irak1, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Santa Cruz Biotechnology (1 mentions)
β actin, by Abcam (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
11 protocols using interleukin 4 (il 4)
1
Differentiation of M1 and M2a Macrophages
2
Macrophage Polarization Modulation
3
In Vitro Antiproliferative Effects of PEG-EV-DOX
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The in vitro antiproliferative effects of PEG-EV-DOX treatment was assessed on B16.F10 melanoma cells in monoculture as well as in co-culture with bone marrow differentiated M2 TAM with 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF, Cell Signaling Technology, MA, USA) and 20 ng/ml interleukin-4 (IL-4, Cell Signaling Technology, MA, USA), as previously described by Rauca et al., 2018.87 (link) For this, ELISA BrdU-colorimetric immunoassay (Roche Applied Science, Penzberg, Germany) was used, as previously described and according to the manufacturer’s instructions.62 (link) Thus, to test the efficacy of PEG-EV-DOX as compared to free DOX, B16.F10 melanoma cells were seeded in a 96-well at a ratio of 5000 cells/well for monocultures, while for co-cultures a ratio of 4000 B16.F10 cells to 1000 M2 TAM/well was used. This cell density ratio (4:1) was reported to approximate the in vivo physiological conditions of murine melanoma development.88 (link) After cells were allowed to attach for 24 h, serial concentrations of PEG-EV-DOX or DOX (ranging between 0.008 and 0.125 µM DOX) were tested in triplicate to assess the IC50 values after 24 h incubation with the treatment. The results were expressed as % of proliferation compared to control (untreated cells in monoculture and, respectively, in co-culture).
4
Protein Expression Analysis in Kidney Samples
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Twenty micrograms of proteins from the kidneys or DC homogenates were separated on 15 or 10% (wt/vol) polyacrylamide denaturing gels and electro-blotted onto polyvinylidene fluoride membranes. The primary antibodies used were anti-caspase-3 (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), IFN-γ (1 : 1000; Cell Signaling Technology), IL-4 (1 : 1000; Cell Signaling Technology), IL-10 (1 : 1000; Cell Signaling Technology), SOCS1-3 (1 : 1000; Cell Signaling Technology), Jak-2 (1 : 1000; Cell Signaling Technology), phospho-STAT3 (1 : 1000; Cell Signaling Technology), NF-κB (1 : 1000; Cell Signaling Technology), TLR-2 (1 : 1000; Cell Signaling Technology), TLR-4 (1 : 1000; Cell Signaling Technology) and IRAK1 (1 : 1000; Cell Signaling Technology). The semiquantitative analysis (AlphaView Software 3.3; ProteinSimple, San Jose, CA, USA) results were expressed as the optical volume densities (OD x mm2) normalized to β-actin (1 : 10 000 dilution; Abcam).
5
Pulmonary Tissue Protein Expression Analysis
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Expression of pulmonary tissue protein was measured by Western blotting as previously described51 (link). Briefly, pulmonary tissues were homogenized in 500 μL of radioimmunoprecipitation assay (RIPA) buffer (EMD Millipore, Billerica, MA, USA). The homogenates were centrifuged at 12,000 rpm for 30 min at 4 °C. The protein (50 μg) was then separated by SDS-PAGE in a 10% polyacrylamide gel and electrotransferred onto a polyvinylidene difluoride membrane. The membranes were incubated in blocking solution (5% BSA) at room temperature for 1 h. The membranes were washed three times (5 min each) with 0.1% T-TBS and then incubated with primary antibody (NLRP3, NF-κB, p-NF-κB, caspase-1, IL-1β, IL-6, TNF-α, IL-4, α-SMA and β-actin; Cell Signaling Technology, Inc., Danvers, MA, USA) in 0.05% T-TBS containing 2.5% BSA at room temperature for 2 h. After washing, the membranes were incubated with peroxidase-conjugated anti-mouse/rabbit antibody (Abcam, Inc., Cambridge, MA, USA) in 0.01% T-TBS at room temperature for 1 h. The membranes were developed with an enhanced chemiluminescence (ECL, Millipore Corporation, Billerica, MA, USA) detection system.
6
Macrophage Polarization and Characterization
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THP-1 monocytes were induced by 100 μM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 48 h to stimulated M0 macrophage differentiation. The M0 macrophages were induced to polarize into M1 macrophages by incubation with lipopolysaccharide (LPS) (100 pg/mL) (Cell Signaling) or into M2 macrophages by IL-4 (20 ng/mL) (Cell Signaling) for another 24 h. Macrophage polarity was confirmed by the mRNA expression levels of F4/80, CD68, CD80, CD86, CD163, CD206, TGF-β, Arginase-1 and IL-1β using quantitative RT-PCR or analyzed the amounts of F4/80, iNOS, p-p38 (Thr180/Tyr182), p38, CD80, TNF-α, CD163, Arginase-1 and IL-10 protein.
7
Isolation and Stimulation of Intestinal Enterocytes
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The small intestine was flushed with ice-cold phosphate buffered saline and cut into 1 cm segments and then transferred into BSS buffer (1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate, 8 mM KH2PO4, 5.6 mM Na2HPO4 and 15 mM EDTA) (isolation buffer), vortexed at 4 °C for 10 minutes, transferred into fresh isolation buffer, and vortexed for another 10 minutes. The crypts and villi were separated via a 70 µm cell strainer, spun down at 500 g for 3 minutes and washed once with ice-cold PBS. For ex vivo stimulation, the enterocytes were resuspended in DMEM/F12 medium and stimulated with 50 ng/ml IL-4 (#5208, Cell Signaling Technology, Danvers MA) or IL-13 (#210-13, PEPROTECH, Rocky Hill NJ) in a tissue culture incubator for 10 minutes. The enterocytes were collected, lysed with sodium dodecyl sulfate sample buffer and analyzed by Western blot assay. For organoid culture, the crypt fraction was mixed with matrigel (Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, #356231, Corning NY) and the standard protocol17 (link) was followed to generate organoids using IntestiCult™ Organoid Growth Medium (Mouse) (#06005, STEMCELL Technology, Cambridge MA). To induce tuft cell differentiation, the organoids were stimulated with 50 ng/ml IL-4 for at least 48 hours. The organoids were then fixed for immunofluorescence, or harvested for protein & mRNA analysis.
8
Astragalus Polysaccharide and Cisplatin in Cancer
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Astragalus polysaccharide (PG2) lyophilized powder obtained from PhytoHealth (PhytoHealth Corporation, Taipei, Taiwan), and cisplatin purchased from Sigma-Aldrich (St Louis, MO, USA), were dissolved in dimethyl sulfoxide (DMSO) to generate 10mM stock solutions. Verapamil, Hoechst 33342, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) dye were also purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against CD80, CD206, NF-κB p65, CD11b, CD31, IL-4, IL-6, IL-10, IL-13, Interferon gamma (IFN-γ), and β-actin were purchased from Cell Signaling Technology (Boston, MA, USA), while human recombinant IL-4, IL-13, IFN-γ, granulocyte-macrophage colony stimulating factor (GM-CSF), and lipopolysaccharide (LPS) were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).
9
Murine BV2 Cell Polarization Protocol
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Murine BV2 cell line, which was originally immortalized after infection with a v-raf/v-myc recombinant retrovirus [16 (link)], and maintained at 37℃ at 5% CO2 at Roswell Park Memorial Institute (RPMI)-1640 (Thermo Scientific) with 10% fetal bovine serum (FBS; Thermo Scientific) and 1% penicillin/streptomycin (Thermo Scientific). To polarize the M1 phenotype activated by LPS or M2 phenotype activated by IL-4, BV2 cells were treated with LPS (20 ng/ml, Sigma-Aldrich) and IL-4(20 ng/ml, cell signaling), respectively. Cultured media containing the BV2 cells were transferred to serum-free RPMI-1640 media after 24 h stimulation and cultured further for 48 h. The serum-free RPMI-1640 conditioned media was collected with a 0.22 µm filter and stored at -70℃ until use [13 (link)]. To determine the role of TGF-α in M2 conditioned media, TGF-α antibody (0.2 ng/ml) was added to the M2 conditioned media for TGF-α neutralization. The control media used serum-free RPMI-1640 media.
10
Cytokine Profiling in Lung Lavage
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Lung lavage was performed with 1 ml saline, and the bronchoalveolar lavage fluid was obtained (approximately 0.8 ml). After centrifugation at 1,500 r/min at 4 °C for 5 min, the supernatant was obtained. Totally 100 μL sample was added into the 96-well plate containing the primary antibodies (IL-1β, IL-4, and IL-5; all from Cell Signaling, Beverly, MA, United States), which was gently shaken and then placed in a water bath at 37 °C for 2 h. The solution was discarded, and the biotin-labeled antibody working solution was added to incubate the plate for 1 h. After washing for 3 times (soaking for 2 min each time), 100 μL horseradish peroxidase-labeled avidin working solution was added, and the plate was heated for 1 h. Then the liquid was discarded, and 90 μL substrate solution was added into each well, which was kept in dark for 15 min. The reaction was stopped by adding 50 μL stop solution. The absorbance at 450 nm was detected within 5 min, and the values were calculated.
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