Splenic lymphocytes were harvested by gradient centrifugation using the lymphocyte separation medium (Dakewe Biotech, China) at room temperature and washed twice with PBS. To determine cytokine expression, cells were stimulated with phorbol12-myristate 13-acetate (PMA)/ionomycin and monensin (Multiscience, China) for 6 h. For flow cytometric analysis, cells were suspended in staining buffer and incubated for 15 min at room temperature by labeling APC/Cy7 anti-mouse CD3 (Clone: 17A2, Biolegend, USA), Brilliant Violet 421TM anti-mouse CD8a (Clone: 53-67, Biolegend, USA), and PE anti-mouse CCL5 (Clone: 2E9, Biolegend, USA). The cells were finally resuspended in 500 μL of PBS and subjected to flow cytometry (BD LSR II).
Monensin
Manufactured by MultiSciences Biotech
Sourced in China
Monensin is a polyether ionophore compound used in laboratory settings. It functions as an ion carrier, facilitating the transport of monovalent cations such as sodium, potassium, and hydrogen across cell membranes. Monensin is commonly utilized in various research applications, including the study of cellular processes and the development of pharmaceutical products.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by MultiSciences Biotech (4 mentions)
Anti il 17 pe, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Brefeldin a, by MultiSciences Biotech (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Apc cy7 anti mouse cd3, by BioLegend (1 mentions)
Lsr 2, by BD (1 mentions)
Lymphocyte separation medium, by Dakewe (1 mentions)
Ifn γ apc, by BioLegend (1 mentions)
Recombinant human il 12, by Thermo Fisher Scientific (1 mentions)
Fix perm kit, by Thermo Fisher Scientific (1 mentions)
Anti foxp3 phycoerythrin pe, by Thermo Fisher Scientific (1 mentions)
Fix perm solution, by Thermo Fisher Scientific (1 mentions)
Cd8 apc efluor 780, by Thermo Fisher Scientific (1 mentions)
Anti cd45 percp cy5, by Thermo Fisher Scientific (1 mentions)
Fixable viability stain 510, by Thermo Fisher Scientific (1 mentions)
Cd206 pecy7, by Thermo Fisher Scientific (1 mentions)
Anti cd45 percp cy5, by BioLegend (1 mentions)
Anti ifn γ apc, by Thermo Fisher Scientific (1 mentions)
F4 80 apc, by BioLegend (1 mentions)
9 protocols using monensin
1
Flow Cytometric Analysis of Splenic Lymphocytes
2
Intracellular IFN-γ Production in PBMCs
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PBMCs were stimulated with recombinant human IL-12 (10 ng/mL; Peprotech, United States) and IL-18 (50 ng/mL; SAB, United States) for 21 h at 37 ˚C, and then 2 µM/L monensin (MultiSciences, China) was added for the final 3 h. Cells were fixed and permeabilized, followed by intracellular staining for IFN-γ-APC (Biolegend, CA, United States).
3
Assessing Immune Modulation in Liver Fibrosis
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To analyze the immune-regulatory effect of DC-IL10 on Th17 and Treg cells in CCl4-induced liver fibrosis, the percentages of Th17 and Treg cells in the mouse spleen were measured by flow cytometry. Splenocytes were isolated from the cell suspensions by gradient centrifugation at 450 × g for 25 min at 4°C with Lymphoprep. Following surface staining with anti-CD4-fluorescein isothiocyanate (FITC, 11-0041; eBioscience, San Diego, CA, USA) and anti-CD25-peridinin-chlorophyll-protein (APC, 45-0251; eBioscience), the cells were fixed, permeabilized, and stained with anti-FoxP3-phycoerythrin (PE, 12-5773; eBioscience) to detect the percentage of Treg cells. For Th17 cell detection, the lymphocytes from the spleen were stimulated for 5 h with 50 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 μg/ml ionomycin, 3 μg/ml brefeldin A (BFA), and 1.4 μg/ml monensin (MULTI SCIENCES, China) in RPMI 1640 containing 10% FBS. The cells were harvested and stained with anti-CD4-FITC then fixed and permeabilized with Fix/PERM kit (eBioscience, USA) and stained intracellularly with anti-IL-17-PE (12-7177; eBioscience). Cells stained with IgG isotype control were used as controls. All antibodies were purchased from eBioscience (USA). Data were obtained using a FACSCalibur flow cytometer and analyzed using FlowJo 7.6.1 software [12 (link)].
4
Immunophenotyping of Th17 and Treg Cells
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The peripheral blood mononuclear cells (PBMC) were isolated from whole blood using the lymphocyte isolation Kit (Hao yang BM, Tianjin, China), and 2 × 106 cells were aliquoted to each tube. For Th17 cells analysis, cells were incubated in RPMI 1640 medium containing 10% FBS (Gibco, USA), 50 ng/mL phorbol 12-myristate 13-acetate (PMA), 1 μg/mL ionomycin, 3 μg/mL brefeldin A (BFA), and 1.4 μg/mL monensin (Multi-Sciences, China) at 37°C with 5% CO2 for 6 hours. Cells were firstly stained with anti-CD4-FITC. Next, the cells were permeabilized using the Fix/Perm solution (eBioscience, USA) and stained with anti-IL-17-PE after fixation. For analysis of Treg cells, cells were firstly stained with anti-CD4-FITC and anti-CD25-APC synchronously and then stained with anti-Foxp3-PE after fixation and permeabilization as described above. All the antibodies used above were purchased from eBioscience (USA) and incubated for 30 min at 4°C in the dark. Finally, cells were resuspended in 1% paraformaldehyde and analyzed using a FACSCalibur (BD, USA) within 1 hour. Appropriate isotype controls and single staining controls were used. The data were analyzed using FlowJo7.6.1 software.
5
Multiparametric Immune Profiling of Tumor
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Single-cell suspensions derived from the tumor, LN, spleen, blood or abdominal cavity were harvested and stained with fluorochrome-conjugated antibodies. However, the tumor single-cell suspensions were stained with Fixable Viability Stain 510 (Thermo Fisher Scientific) prior to antibody staining to distinguish between living and dead cells. To analyze M2 macrophages, cells were stained with anti-CD45 PerCP/Cy5.5 (BioLegend), F4/80 APC (BioLegend), CD11b PE (BioLegend) and CD206 PE/CY7 (eBioscience) antibodies. To detect CD8+CD122+ Tregs, cells were stained with anti-CD45 PerCP/Cy5.5, CD8 APC/eFluor780 (eBioscience) and CD122 PE or isotypes (BioLegend) antibodies. To measure IFN-γ+ T cells, 1 × 106 cells were incubated in 96-well plates in the presence of PMA (50 ng/ml, MultiSciences) and Ionomycin (1 μg/ml, MultiSciences) for 6 h at 37°C. Monensin (5 μg/ml, MultiSciences) was added 2 h after the addition of PMA/Ionomycin. Cells were first stained with anti-CD45 PerCP/Cy5.5 and CD8 APC/eFluor780, fixed in 1% paraformaldehyde, permeabilized and then stained with anti-IFN-γ APC (eBioscience). Cells finally were analyzed through FACSCalibur (BD Biosciences).
6
PBMC Isolation and Stimulation for AD
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Peripheral blood samples were collected from AD patients and healthy controls, and the PBMCs were prepared by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Stockholm, Sweden). Cell suspensions were adjusted to a concentration of 5×106/mL and then incubated for 5 hours at 37°C in a 5% CO2 incubator, with or without phorbol myristic acid (PMA, 50 ng/mL)/ionomycin calcium (1μg/mL) mixture and brefeldin A (3μg/mL)/monensin (1.4 μg/mL) mixture (MultiSciences Biotech Co Ltd, Hangzhou, China) dissolved in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Grand Island, NY).
7
Flow Cytometric Analysis of Th17 and Treg Cells
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The proportions of Th17 and Treg cells were detected using flow cytometry. In brief, lymphocytes were isolated from the spleens of mice and washed twice by phosphate-buffered saline (PBS) solution. Subsequently, the cells were transferred to test tubes and incubated with surface antibody of FITC-labeled CD4 (rat anti-mouse, BD, 553046, USA) and APC-labeled CD25 (rat anti-mouse, BD, 557192, USA). After being washed again by PBS, 1 ml of 1640 medium (Gibco, USA) containing phorbol 12-myristate 13-acetate (PMA, MultiSciences, China), lonomycin (MultiSciences, China), and monensin (MultiSciences, China) was added in each tube, incubated at 37°C for 4 h, and washed again. Next, cells were fixed and permeated successively, followed by incubating with intracellular antibody, including PE-labeled IL-17A (rat anti-mouse, BD, 559502, USA), PE-labeled retinoic acid−related orphan receptor γt (RORγt, rat anti-mouse, ebioscience, 12-6981-60, USA), and PE-labeled FoxP3 (rat anti-mouse, BD, 563101, USA), at a temperature of 4°C for 60 min. After being washed again and analyzed by a FACSCalibur flow cytometer (Beckman Coulter, USA), the resulting data were processed by FlowJo software (Tree Star, Ashland).
8
Identification and Analysis of B10 Cells
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The fluorescent mAbs CD4, CD19, CD11c, F4/80, CD1d, CD5 and IL-10 were purchased from Elabscience (Wuhan, China). Intracellular staining was conducted as described previously (Yang et al., 2012 (link)). Splenocytes from mice were suspended in the presence of PMA/ionomycin mixture (Phorbol 12-myristate 13-acetate, Multisciences) and monensin (Multisciences) for 5 hours to analyze B10 cells (CD1dhiCD5+B19+IL-10+). Then, the cells were collected and stained with PE Anti-CD19 mAbs, FITC anti-CD1d mAbs and PerCP/Cyanine5.5 anti-CD5 mAbs. After removing the unbound antibodies, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences, San Jose, CA). Then, they were stained with APC mouse anti-IL-10 following the manufacturer’s instructions. The samples were performed using BD FACSCanto flow cytometer (BD Biosciences), and the results were analyzed using FlowJo Software (Version X; TreeStar, Ashland, OR).
9
Intracellular Cytokine Profiling by Flow Cytometry
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Intracellular cytokines were studied by flow cytometry to identify the cytokine-producing cells. For intracellular cytokine staining, treated cells suspended with 140 μL RPMI-1640 medium was incubated for 4 h at 37°C in 5% CO2 in the presence of 25 ng/mL of phorbol myristate acetate (PMA), 1 μg/mL of ionomycin, and 1.7 μg/mL of monensin (all from Multi Sciences, China). After incubation, the cells were stained with PE-Cy5-conjugated anti-CD3 and fluorescein isothiocyanate-conjugated anti-CD8 monoclonal antibodies at room temperature in the dark for 15 min to delimit CD4+ T cells because CD4 was downmodulated when cells were activated by PMA. After surface staining, the cells were stained with PE-conjugated anti-IL-17 and Fluor 660-anti-human IL-22 monoclonal antibodies after fixation and permeabilization according to the manufacturer's instructions. Stained cells were analyzed by flow cytometric analysis using a Beckman Gallios cytometer equipped with the Kaluza software (Beckman Coulter, USA).
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