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Mil 33

Manufactured by R&D Systems
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Sourced in United Kingdom

The MIL-33 is a laboratory equipment designed for performing various experimental tasks. It features a durable construction and precise controls to enable consistent and reliable operation. The core function of the MIL-33 is to facilitate controlled experimentation and data collection across a range of scientific disciplines.

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Lab products found in correlation

Mil 7, by R&D Systems (3 mentions) Mil 6, by R&D Systems (2 mentions) Recombinant mil 1β, by R&D Systems (2 mentions) Mil 36γ, by R&D Systems (2 mentions) Recombinant hil 1β, by R&D Systems (2 mentions) Steady glo, by Promega (2 mentions) M1000 plate reader, by Tecan (2 mentions) Fluorochrome conjugated streptavidin, by BD (2 mentions) Mil 25, by R&D Systems (2 mentions) Hil 6, by R&D Systems (1 mentions) Htnf α, by R&D Systems (1 mentions) Regiiiγ, by Cloud-Clone (1 mentions) Specific taqman probes, by Thermo Fisher Scientific (1 mentions) Qscript cdna synthesis kit, by Quanta Biosciences (1 mentions) Abi 7500 thermal cycler, by Thermo Fisher Scientific (1 mentions) Rneasy kit, by Qiagen (1 mentions) Recombinant mouse il 2, by R&D Systems (1 mentions) P38 inhibitor sb203580, by Merck Group (1 mentions) Klrg1 2f1, by Thermo Fisher Scientific (1 mentions) Mtgf β1, by R&D Systems (1 mentions)

9 protocols using mil 33

1

Cytokine Profiling in Skin and Cell Cultures

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The supernatants of skin homogenate or cell culture supernatants were collected for cytokine evaluation. The samples with low yield of protein were pre-determined and excluded. Cytokine production was measured by ELISA kits of mTNF-α (BD Biosciences), hTNF-α (R&D systems), mIL-6 (R&D systems), hIL-6 (R&D systems), mIL-17 (R&D systems), mIL-33 (R&D systems) and RegIIIγ (Cloud Clone corp.) according to the manufacturer's instructions.
Wu Y., Quan Y., Liu Y., Liu K., Li H., Jiang Z., Zhang T., Lei H., Radek K.A., Li D., Wang Z., Lu J., Wang W., Ji S., Xia Z, & Lai Y. (2016). Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes. Nature Communications, 7, 13393.
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2

Antagonism of IL-1, IL-33, and IL-36 Signaling

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The functionality of MAB-mR3 was tested in murine NIH-3T3 (NF-κB) Luciferase reporter cell lines (Signosis, DMEM, 10% FCS). Seeded in 384-well flat bottom plates (20,000 cells/well (30,000 cells/well for IL-33 stimulations)) and rested for 16hrs (37°C/5% CO2) before pre-incubating 1hr with MAB-mR3 antibody at increasing concentrations. Recombinant mIL-1β (50pg/mL), mIL-33 (1ng/mL) or mIL-36γ (170ng/mL) (R&D Systems) was added and plates incubated for 5hrs (37°C/5% CO2). Steady-Glo™ (Promega) solution was used on lysates and luminescence measured (Tecan M1000 plate reader). Inhibition of IL-6 release was determined in a NIH-3T3 cell line (ATCC). Seeded in 384-well flat bottom plates (12,500 cells/well) and rested for 2hrs (37°C/5% CO2) before pre-incubating 1hr with MAB-mR3 or MAB-hR3 antibody at increasing concentrations. Cells were stimulated with recombinant hIL-1β (50pg/mL, R&D Systems) for 16hrs and assayed for IL-6 production.
Højen J.F., Vindvad Kristensen M.L., McKee A.S., Wade M.T., Azam T., Lunding L.P., de Graaf D.M., Swartzwelter B.J., Wegmann M., Tolstrup M., Beckman K., Fujita M., Fischer S, & Dinarello C.A. (2019). IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease. Nature immunology, 20(9), 1138-1149.
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3

Transcriptomic analysis of mast cell activation

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BMBs were activated with either IgE/DNP or 10 ng/ml mIL-33 (R&D Systems) for 4 hours. mRNA was extracted by RNeasy kit (Qiagen) and cDNA was generated by qScript cDNA synthesis kit (Quanta BioSciences). Microarray assessment was performed by the Northwestern University Genomics Core using llumina MouseWG-6 Beadchips. Heatmaps were generated using GENE-E software (Broad Institute, http://broadinstitute.org/cancer/software/GENE-E). Network diagrams were generated using the “FGNet” R package and the GeneTerm Linker algorithm (adjusted P value < 0.05; minimum support of 3). Visualization of these networks was performed with the “iGraph” R package and Cytoscape 3.2.1. Gene expression was also determined by real-time PCR using an ABI 7500 thermal cycler (Applied Biosystems) and specific TaqMan probes (Applied Biosystems) for each gene of interest. β-actin expression was used as an internal control, and changes in the threshold cycle values were determined.
Hsu C.L., Chhiba K.D., Krier-Burris R., Hosakoppal S., Berdnikovs S., Miller M.L, & Bryce P.J. (2020). Allergic inflammation is initiated by IL-33–dependent crosstalk between mast cells and basophils. PLoS ONE, 15(1), e0226701.
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4

Multiparameter Flow Cytometry Phenotyping

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mAbs specific for mouse B220 (RA3-6B2), c-Kit (2B8), CD3ε (145-2C11), CD5 (53-7.3), CD8 (53-6.7), CD11b (M1/70), CD11c (HL3), CD16/CD32 (2.4G2), CD19 (1D3), CD25 (PC61), CD34 (RAM34), CD45.1 (A20), CD45.2 (104), CD49b (DX5), CD127 (A7R34), GATA3 (L50-823), Gr-1 (RB6-8C5), MHC class II (M5/114.1), NK1.1 (PK136), Sca-1 (D7), Siglec-F (E50-2440), ST2 (U29-93), Thy1.2 (53-2.1), IL-4 (11B11), IL-12 (C15.6), IFN-γ (XMG1.2), and fluorochrome-conjugated streptavidin were purchased from BD Biosciences. mAbs specific for mouse CD4 (GK1.5), Flt3 (A2F10), F4/80 (BM8), IL-5 (TRFK5), IL-17RB (9B10), and NKp46 (29A1.4) were purchased from BioLegend. mAbs specific for mouse α4β7 (DATK32), FcεRIα (MAR-1), IL-13 (eBio13A), and killer cell lectin-like receptor G1 (KLRG1; 2F1) were purchased from eBioscience. mAbs against mouse CD16/CD32 (2.4G2), CD28 (37.51), and erythroid cell marker (TER-119) were purified from hybridoma culture supernatants in our laboratory.
Recombinant mouse IL-2, mIL-4, mIL-6, mIL-7, mIL-33, and mTGF-β1 were purchased from R&D Systems. p38 inhibitor (SB203580) and actinomycin D were purchased from Sigma-Aldrich.
Hikichi Y., Motomura Y., Takeuchi O, & Moro K. (2021). Posttranscriptional regulation of ILC2 homeostatic function via tristetraprolin. The Journal of Experimental Medicine, 218(12), e20210181.
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5

Multiparametric Flow Cytometry Analysis of Mouse Immune Cell Subsets

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For flow-cytometric analyses, mAbs specific for mouse CD3ε (145-2C11), CD8α (53–6.7), CD19 (1D3), B220 (RA3-6B2), NK1.1 (PK136), CD11b (M1/70), CD11c (HL3), Gr-1 (RB6-8C5), TER119 (TER119), CD45.1 (A20), CD45.2 (2F1), Sca-1 (E13-161.7), CD25 (PC61), Thy1.2 (53–2.1), Flt3 (A2F10), α4β7 (DATK32), KLRG1 (2F1), CCR9 (CW-1.2), CD31 (MEC13.3), GATA3 (L50-823), T-bet (O4-46), RORγt (Q31-378), IFN-γ (XMG1.2), IL-17A (TC11-18H10), and fluorochrome-conjugated streptavidin were purchased from BD. mAbs against mouse Notch1 (HMN1-12), Notch2 (HMN2-35), CD4 (GK1.5), PDGFRα (ATA5), gp38 (8.1.1), and PLZF (9E12) were purchased from BioLegend. mAbs against c-Kit (2B8), FcεRIα (MAR-1), IL-7Rα (A7R34), and IL-13 (eBio13A) were purchased from eBioscience. Anti-T1/ST2 (DJ8) was purchased from MD Biosciences. mAb against mouse CD16/CD32 (2.4G2) was purified from hybridoma culture supernatants in our laboratory.
Recombinant mIL-2, mIL-7, mIL-25, mIL-33, and mTSLP were purchased from R&D Systems. A STAT5 inhibitor (CAS 285986–31-4) was purchased from Calbiochem, and Dox was purchased from Clontech.
Koga S., Hozumi K., Hirano K.I., Yazawa M., Terooatea T., Minoda A., Nagasawa T., Koyasu S, & Moro K. (2018). Peripheral PDGFRα+gp38+ mesenchymal cells support the differentiation of fetal liver–derived ILC2. The Journal of Experimental Medicine, 215(6), 1609-1626.
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6

Antagonism of IL-1, IL-33, and IL-36 Signaling

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The functionality of MAB-mR3 was tested in murine NIH-3T3 (NF-κB) Luciferase reporter cell lines (Signosis, DMEM, 10% FCS). Seeded in 384-well flat bottom plates (20,000 cells/well (30,000 cells/well for IL-33 stimulations)) and rested for 16hrs (37°C/5% CO2) before pre-incubating 1hr with MAB-mR3 antibody at increasing concentrations. Recombinant mIL-1β (50pg/mL), mIL-33 (1ng/mL) or mIL-36γ (170ng/mL) (R&D Systems) was added and plates incubated for 5hrs (37°C/5% CO2). Steady-Glo™ (Promega) solution was used on lysates and luminescence measured (Tecan M1000 plate reader). Inhibition of IL-6 release was determined in a NIH-3T3 cell line (ATCC). Seeded in 384-well flat bottom plates (12,500 cells/well) and rested for 2hrs (37°C/5% CO2) before pre-incubating 1hr with MAB-mR3 or MAB-hR3 antibody at increasing concentrations. Cells were stimulated with recombinant hIL-1β (50pg/mL, R&D Systems) for 16hrs and assayed for IL-6 production.
Højen J.F., Vindvad Kristensen M.L., McKee A.S., Wade M.T., Azam T., Lunding L.P., de Graaf D.M., Swartzwelter B.J., Wegmann M., Tolstrup M., Beckman K., Fujita M., Fischer S, & Dinarello C.A. (2019). IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease. Nature immunology, 20(9), 1138-1149.
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7

ILC2 Cell Proliferation Assay

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Two thousand sorted stomach or SI ILC2s (CD45 + CD3 -CD19 -TCRb -CD127 + Sca1 + KLRG1 + IL-33R + cells) were cultured in the presence of recombinant mIL-2, mIL-7, mIL-25 or mIL-33 (10 ng/ml each; R&D systems) for 6 days, and cell proliferation were determined using the CellTiter-GloÒ Luminescent Cell Viability Assay (Promega) and a luminometer (Promega). The baseline was established from two thousand cells of sorted stomach or SI ILC2s without culture or stimulation. Serial dilutions of fresh splenocytes were used to obtain a standard curve and cell numbers were calculated following the formula based on this standard curve.
Satoh-Takayama N., Kato T., Motomura Y., Kageyama T., Taguchi-Atarashi N., Kinoshita-Daitoku R., Kuroda E., Di Santo J.P., Mimuro H., Moro K, & Ohno H. (2020). Bacteria-Induced Group 2 Innate Lymphoid Cells in the Stomach Provide Immune Protection through Induction of IgA. Immunity, 52(4).
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8

Quantification of Growth Factors and Cytokines

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Tissue and in vitro cell samples lysed with a lysis buffer (Cat. No. 3228, Sigma), followed by 20-min centrifugation to remove cellular debris. Plasma samples were obtained from whole blood processed by collection into anti-coagulant-containing plasma tubes followed by 15-min centrifugation. Conditional media were collected at 48 or 72 h of confluent monolayer cells and were centrifuged to remove cellular debris before use. Four different assays were performed according to manufacturer's protocol to detect hPDGF-BB (Cat. No. DY220, R&D Systems), mPDGF-BB (Cat. No. MBB00, R&D Systems), mIL-33 (Cat. No. M3300, R&D Systems) or hIL-33 (Cat. No. 435907, BioLegend). Optimal standard curves were applied to individual assays and the absorbance values were detected at 450 nm using a microplate reader.
Yang Y., Andersson P., Hosaka K., Zhang Y., Cao R., Iwamoto H., Yang X., Nakamura M., Wang J., Zhuang R., Morikawa H., Xue Y., Braun H., Beyaert R., Samani N., Nakae S., Hams E., Dissing S., Fallon P.G., Langer R, & Cao Y. (2016). The PDGF-BB-SOX7 axis-modulated IL-33 in pericytes and stromal cells promotes metastasis through tumour-associated macrophages. Nature Communications, 7, 11385.
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9

Recombinant IL-33 Characterization and Validation

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The recombinant human IL-33 (hIL-33), provided by our collaborators in Glasgow with a purity of more than 95%, had been routinely tested for its endotoxin (LPS) levels (<0.01 EU/μg) by the Limulus amebocyte lysate QCL-1000 pyrogen test (BioWhittaker). Moreover, the bioactivity and specificity of our recombinant hIL-33 were also further confirmed by its ability to induce IL-5 production using Th2 cells polarized from WT and IL-33 receptor (IL-33R) knock-out (ST2−/−) mice (ref.30). The recombinant mouse IL-33 (mIL-33) was obtained commercially with a purity of more than 98%, and an endotoxin level less than 1.0 EU/μg, according to the supplier (R&D Systems Inc., UK).
Sattler S., Ling G.S., Xu D., Hussaarts L., Romaine A., Zhao H., Fossati-Jimack L., Malik T., Cook H.T., Botto M., Lau Y.L., Smits H.H., Liew F.Y, & Huang F.P. (2014). IL-10-producing regulatory B cells induced by IL-33 (BregIL-33) effectively attenuate mucosal inflammatory responses in the gut. Journal of Autoimmunity, 50(100), 107-122.
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