Splenocytes were isolated from spleens. For Treg cell staining and detection, antimouse CD4 fluorescent monoclonal antibody labeled with fluorescein isothiocyanate (eBioscience, San Jose, CA, USA) and antimouse CD25 labeled with allophycocyanin (eBioscience) were used as surface markers. After washing with PBS, cells were fixed, permeabilized, and labeled with phycoerythrin-conjugated antimouse FoxP3 antibody (eBioscience). For Th17 cell analysis, cells were incubated with Phorbol-12-myristate-13-acetate (PMA) (50 ng/mL), ionomycin (1 μg/mL), and monesin (1.7 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 6 hours to activate T cells and stimulate the accumulation of intracellular IL-17. Surface staining was performed with fluorescein isothiocyanate-conjugated antimouse CD4 antibody. Cytofix/Cytoperm reagent (BD Biosciences, San Jose, CA, USA) was used to fix and permeabilize cells. Cells were stained with phycoerythrin-conjugated antimouse IL-17A antibody (eBioscience). Stained cells were analyzed by flow cytometry on an FACS Calibur flow cytometer (BD Biosciences). The frequency of CD4+CD25+FoxP3+Tregs and CD4+IL17+Th17 cells was analyzed using FlowJo software (BD Biosciences).
Monesin
Manufactured by Merck Group
Sourced in United States
Monensin is a polyether ionophore antibiotics produced by the bacterium Streptomyces cinnamonensis. It functions as an antibiotic and coccidiostat.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (3 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Lincomycin, by Merck Group (2 mentions)
Sulfamethoxazole, by Merck Group (2 mentions)
Sulfamerazine, by Merck Group (2 mentions)
Griseofulvin, by Merck Group (2 mentions)
Norfloxacin, by Merck Group (2 mentions)
Azithromycin, by Merck Group (2 mentions)
Oxytetracycline, by Merck Group (2 mentions)
Sulfadiazine, by Merck Group (2 mentions)
Spectinomycin, by Merck Group (2 mentions)
Florfenicol, by Merck Group (2 mentions)
Cefuroxime, by Merck Group (2 mentions)
Sulfadimethoxine, by Merck Group (2 mentions)
Sarafloxacin, by Merck Group (2 mentions)
Difloxacin, by Merck Group (2 mentions)
Tylosin, by Merck Group (2 mentions)
Sulfathiazole, by Merck Group (2 mentions)
Sulfaquinoxaline, by Merck Group (2 mentions)
Sulfamethoxypyridazine, by Merck Group (2 mentions)
10 protocols using monesin
1
Murine Treg and Th17 Cell Analysis
2
Cytokine Expression in Antigen-Specific T Cells
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Freshly isolated splenocytes or lymphocytes were plated into round-bottom 96-well plates (2 × 106 cells per well) and stimulated with 5 μg/mL OVA peptide: OVA(257–264) and OVA(323–339) and ENV peptide pool: ENV(B strain, RL 42). 1 h later, brefeldin A (Cell Signaling Technology Cat#9972S) and monesin (Sigma Cat#1445481) were added to each well at final concentrations of 1 μg/mL and 1 μmol/L. Then 7 h later, the stimulation were stopped by washing the plates with R10 medium and 4 cell surface markers (CD3, CD8, CD44, and CD62L) were stained on ice with uorescein labeled antibodies, including PerCP-Cy5.5-labeled anti-mouse CD3 (Clone: 17A2, Biolegend Cat# 100218), Pacic Blue labeled anti-mouse CD8 (Clone: 53-6.7, Biolegend Cat# 100725), FITC-labeled anti-mouse CD44 (Clone: IM7, eBioscience Cat# 11-0441-81), and APC-eFluor780-labeled anti-mouse CD62L (Clone: MEL-14, eBioscience Cat# 47-0621-80). Next, the cells were fixed and permeablized, and intracellular IFN-γ was stained with PE-conjugated anti-mouse IFN-γ (Clone: XMG1.2, Biolegend Cat# 505808). Stained samples were measured using BD FACS Aria I. Data analysis was done by using FlowJo X software (Tree Star, Inc).
3
Isolation and Activation of Murine Naïve CD4+ T Cells
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Splenocytes and lymphocytes were isolated from the spleens and lymph nodes of 4 WT and 4 ERAP1−/− mice. Cells from both tissues were processed into a homogenous single-cell suspension and combined. Naïve CD4+ T cells were isolated from this cell mixture using the MACS Mouse Naïve CD4+ T cell Isolation Kit (Miltenyi Biotec) per manufacturer’s instructions. Cells were plated in complete RPMI medium at 1 × 105 cells/well in a 96-well high binding plate pre-coated with 2 µg/mL anti-CD3 mAb (BioLegend) and 2 µg/mL anti-CD28 mAb (BioLegend). IL-27 (Peprotech) was added to indicated wells at 25 ng/mL. Supernatant was collected from cultures at 96 hours at which point the media was replaced with media containing 0.1 µg/mL PMA (Calbiochem), 1 µg/mL Ionomycin (Calbiochem), 0.2 µL/well of GolgiPlug (BD Biosciences) and 1.95 µM Monesin (Sigma Aldrich). After 5 hours the cells were collected, washed and stained.
4
Comprehensive Analysis of Pharmaceutical Compounds
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Amoxicillin, azithromycin, cefuroxime, chloramphenicol, chlortetracycline, ciprofloxacin, clarithromycin, colistin, danafloxacin, decoquinate, dexamethasone, diclofenac, difloxacin, doxycycline, enrofloxacin, erythromycin, florfenicol, flumethasone, griseofulvin, ibuprofen, levofloxacin, lincomycin, maduramicin, mefenamic acid, monesin, narasin, nicarbazin, norfloxacin, oxytetracycline, paracetamol, propranolol, robenidine, sarafloxacin, salinomycin, spectinomycin, sulfachloropyridine, sulfadiazine, sulfadimethoxine, sulfamerazine, sulfamethasone, sulfamethoxazole, sulfamethoxypyridazine, sulfapyridine, sulfaquinoxaline, sulfathiazole, tetracycline, trimethoprim, and tylosin with a purity above 98% were bought from Sigma-Aldrich (St. Louis, MO, USA). Anhydrous citric acid, trichloroacetic acid (TCA), ethylenediaminetetraacetic acid disodium salt (EDTA), and disodium hydrogen phosphate dehydrate were also purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN), methanol (MeOH) (HPLC grade ≥ 99%), and formic acid (purity > 99% for analysis) were obtained from Acros Organics (Geel, Belgium). Purified water, with a resistivity higher than 18.0 MU, was prepared in the laboratory with a Milli-Q system from Millipore (Burlington, MA, USA).
5
Multidimensional Immune Profiling
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After propagation, the BMDCs and MoDCs were collected for surface co-stimulatory molecules staining. The IDO staining was performed after DCs treated by Fix/Perm Buffer Set (ebioscience, CA, USA). For cytokines intracellular staining, the cells were obtained from the coculture medium and were stimulated with 20 ng/ml PMA and 1 μg/ml ionomycin (Sigma-Aldrich, Shanghai, China) plus 2 μm of Monesin (Sigma-Aldrich, Shanghai, China) for 4 h following fluorescence-conjugated anti-CD4 antibody staining. IFN-γ and Foxp3 staining was performed in accordance with the manufacturer’s instructions (ebioscience, CA, USA).
6
Flow Cytometry Analysis of Treg and Th17 Cells
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PBMCs (7 × 105 cells) were stained with certain antibodies against L/D-FVS510, CD45-FITC, CD4-PE, CD25-BV421 (all from Biolegend, United States) to analyse the Treg frequency, and FOXP3-APC (EB). The impacted cells were run with BD FACSCalibur and BD FACSAria III flow cytometers (BD Biosciences, United States). Using positive gates and gating controls by Fluorescence minus one (FMO) controls. For Th17 cell analysis, cells were incubated with phorbol-12-myristate-13-acetate (PMA) (50 ng/mL), ionomycin (1 μg/mL), and monesin (1.7 μg/mL; Sigma-Aldrich, United States) at 37°C for 6 h to activate T cells and stimulate the accumulation of intracellular IL-17. Then, the cells were stained with specific antibodies against L/D-FVS510, CD45-FITC, CD4-PE (all from Biolegend, San Diego, California, United States), and IL-17A-APC (EB). The FACSCalibur flow cytometer (BD Biosciences) was used to analyse the stained cells.
7
Quantifying MMP-9 in Neutrophils
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To determine the matrix metalloproteinase-9 (MMP-9) production by neutrophils, isolated neutrophils (1 × 106) from healthy controls were stimulated either with RA SF (cell-free SF, DAS-28 score > 5, 1:9 ratio SF:media), rIL-9 (20 ng/ml) or anti-IL-9 blocking antibody (10 μg/ml, Abcam) for 12 hours and monesin was added (as a golgi transport inhibitor, Sigma-Aldrich) during last 6 hours of culture. Neutrophils were fixed with 2% formaldehyde, permeabilized and incubated with rabbit anti-human anti-MMP-9 primary antibody (1:250 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) for 30 minutes at room temperature followed by secondary anti-rabbit IgG FITC (dilution 1:100; Abcam) for another 30 minutes at room temperature. Stained samples were acquired by FACSCalibur (BD) and analyzed with Flow Jo (Tree Star).
8
Th1 Cell Characterization in Mice
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Cells were collected and stimulated with PMA (25 ng/mL) and ionomycin (1 μg/mL) in the presence of 1 μg/mL monesin (Sigma-Aldrich) for 4 hours. Cells were then fixed, permeabilized by using Fixation/Permeabilization Solution Kit (eBioscience) according to the manufacturer's instructions, and stained with FITC-conjugated anti-IFN-γ, PE-conjugated anti-phospho-STAT4, or PE-cy7-conjugated anti-T-bet (eBioscience). In order to stain Th1 cells in mice, single cell suspension was deprived from spleen of WT and Gnaq−/− chimeric mice, stimulated with PMA, ionomycin, and monensin for 4 hour. After culture, cells were stained with PE-conjugated anti-CD4, followed by intracellular staining with FITC-conjugated anti-IFN-γ. Cells were analyzed on Cytomic FC500 (Beckman Coulter), and data were analyzed using FlowJo (Tree Star).
9
Quantification of Veterinary Drugs and Antibiotics
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Amoxicillin, azithromycin, cefuroxime, chloramphenicol, ciprofloxacin, clarithromycin, colistin, danafloxacin, decoquinate, dexamethasone, diclofenac, difloxacin, doxycycline, enrofloxacin, erythromycin, florfenicol, flumethasone, griseofulvin, ibuprofen, levofloxacin, lincomycin, maduramicin, mefenamic acid, monesin, narasin, nicarbazin, norfloxacin, oxytetracycline, paracetamol, propranolol, robenidine, sarafloxacin, salinomycin, spectinomycin, sulfachloropyridine, sulfadiazine, sulfadimethoxine, sulfamerazine, sulfamethasone, sulfamethoxazole, sulfamethoxypyridazine, sulfapyridine, sulfaquinoxaline, sulfathiazole, tamoxifen, tetracycline, trimethoprim and tylosin with purity between 98 and 101% were bought from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN) and methanol (MeOH) (HPLC grade ≥ 99%) were obtained from Acros Organics (Geel, Bergium) and purified water was prepared in the laboratory with a Milli-Q system from Millipore (Burlington, MA, USA).
10
Antigen-specific CD8+ T cell Responses
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Peptide- or antibody-loaded iDCs and mDCs were cultivated with allogeneic expanded CMVNLV- and mNPM1CLA-specific T cells in a 1:5 ratio for 4–6 h in DC medium containing 25 µM monesin and 10 µg/ml of brefeldin A (both Sigma-Aldrich) at 37°C and 5% CO2. Viable Zombie Green-negative (BioLegend) cells were stained for CD8 (PerCP-eFluor710, SK1, eBioscience), subsequently fixed and permeabilized using the BD Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s instructions followed by intracellular IFN-γ (PE, B27) and TNF-α (APC, Mab11, both BioLegend) staining. The percentage of cytokine-secreting CD8+ T cells was determined by flow cytometry.
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