106 cells have been taken from mashed MLN filtrates. Treg cells have been stained for CD4, CD32 and FoxP3 using a mouse regulatory T cell Staining Kit 1 (eBioscience). Cell samples have been run through flow cytometry (BD Accuri) and double positive cells for CD32 and FoxP3 among CD4 positive cells have been counted.
Mouse regulatory t cell staining kit 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mouse Regulatory T Cell Staining Kit #1 is a laboratory product designed for the identification and analysis of regulatory T cells in mouse samples. It contains the necessary reagents and antibodies for the staining and flow cytometric detection of regulatory T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Apc anti mouse cd206, by BioLegend (2 mentions)
Fitc anti mouse f4 80, by BioLegend (2 mentions)
Apc anti mouse cd8a, by BioLegend (2 mentions)
Pe cy7 anti mouse cd4, by BioLegend (2 mentions)
Apc anti mouse gr 1, by BioLegend (2 mentions)
Pe anti mouse cd11b, by BioLegend (2 mentions)
Liberase, by Roche (2 mentions)
Pe anti mouse ki67, by BioLegend (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
7 protocols using mouse regulatory t cell staining kit 1
1
Quantification of Murine Regulatory T Cells
2
Quantification of Tumor-Associated Immune Cells
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Tumor tissues, excised from euthanized mice, were minced and digested using liberase (Roche) for 2 h. The cells were washed and counted, followed by extracellular staining with antibodies, such as APC anti-mouse Gr-1 (Biolegend), PE anti-mouse CD11b (Biolegend), FITC anti-mouse F4/80 (Biolegend), and APC anti-mouse CD206 (Biolegend), to analyze MDSCs and TAMs in the tumor. For analysis of the tumor-infiltrated proliferative T cells, the cells were washed, surface-stained using PE-Cy7 anti-mouse CD4 (Biolegend), and APC anti-mouse CD8a (Biolegend), fixed, and permeabilized, followed by intracellular staining using PE anti-mouse Ki67 (Biolegend). Cells were washed and analyzed using flow cytometry. Detection of Tregs (regulatory T cells) was performed using Mouse Regulatory T Cell Staining Kit#1 (eBioscience), according to manufacturer’s instructions.
3
Splenic Lymphocyte Isolation and Subset Analysis
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The spleens of mice were removed, and the blood was washed off with PBS solution. They were cut into small pieces, ground with a 2 mL syringe head, filtered through a 200-mesh sieve, centrifuged at 400g at 4°C for 5 minutes, and resuspended to obtain a splenic single lymphocyte suspension. Treg and Th17 cells were labeled using the Mouse Regulatory T-cell Staining Kit #1 (eBioscience) and IL-17A monoclonal antibody (eBio17B7), PerCP-Cyanine5.5, according to the manufacturer's instructions. The results were detected by using BD FACSaria II flow cytometer and analyzed using FlowJo v10.
4
Evaluating Th17 and Treg Responses to rTsPmy
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BALB/c mice were divided into 3 groups with 4 mice each, and each group was immunized intraperitoneally with 100 μg of rTsPmy or the same amount of Sf9 insect cell protein as a non-relevant protein control twice at 2 weeks intervals. Another group of 4 mice were given PBS only. Fourteen days after the final immunization, all mice were sacrificed and the splenocytes were harvested for the analysis of cytokine production and the presence of Th17 cells and Tregs.
For FACS analysis of Th17 cells, the harvested splenocytes were stimulated with 25 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA), 1 μg/ml ionomycin (Sigma-Aldrich, St. Louis, MO, USA) and 0.66 μl/ml Golgistop™ (BD Biosciences, San Jose, CA, USA) for 6 h before cell staining with anti-IL-17A-PE-Cyanine7 (eBioscience, San Diego, CA, USA). The culture supernatants were recovered for measuring cytokine release as described above. For detection of Tregs, the harvested splenocytes were directly stained with Mouse Regulatory T Cell Staining Kit #1 according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA).
For FACS analysis of Th17 cells, the harvested splenocytes were stimulated with 25 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA), 1 μg/ml ionomycin (Sigma-Aldrich, St. Louis, MO, USA) and 0.66 μl/ml Golgistop™ (BD Biosciences, San Jose, CA, USA) for 6 h before cell staining with anti-IL-17A-PE-Cyanine7 (eBioscience, San Diego, CA, USA). The culture supernatants were recovered for measuring cytokine release as described above. For detection of Tregs, the harvested splenocytes were directly stained with Mouse Regulatory T Cell Staining Kit #1 according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA).
5
Treg Cell Ratio Analysis in Mouse Blood
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Peripheral blood was taking from canthus vein and kept in EP tubes with heparin sodium on ice before staining. To analyze the ratio of Treg cells in the blood, we used Mouse Regulatory T Cell Staining Kit 1# (eBioscience) according to manufacturer’s protocol. Briefly, after fixation/permeabilization and washing, cells were blocked with anti-mouse CD16/CD32 for 15 min, followed by staining for PE-FoxP3, FITC-CD4, and APC-CD25 for 30 min. After washing twice, samples were collected and analyzed by FACSCalibur (Becton Dickinson Corporation, USA).
6
Immune Cell Profiling in Tumors
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The frequency of different immune cell types was determined using FACS. Myeloid-derived suppressor cells (MDSCs) in tumor or spleen tissue were analyzed using a mouse MDSC Flow Cocktail 1 (CD11b PE/Gr-1 APC/Ly-6G FITC, BioLegend). Regulatory T cells were analyzed using the Mouse Regulatory T Cell Staining Kit #1 (eBioscience). FITC anti-mouse CD8α antibody (clone: 53-6.7, BioLegend), APC anti-mouse CCR5 antibody (clone: HM-CCR5, BioLegend), PE anti-mouse CCR6 antibody (clone: 29-2L17, BioLegend), and PE/Cy7 anti-mouse CCR7 antibody (clone: 4B12, BioLegend) were used to label isolated CD11c+ cells (38 (link)).
7
Comprehensive Immune Profiling of Tumor Microenvironment
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Mice were euthanized, and their tumor tissues and main organs were removed. One gram of tissue was taken from each group of tumors and placed in serum-free 1640 medium for extraction of cells from tumor tissues. First, the tumor tissue was cut into small pieces, Liberase (Roche) was added to a final concentration of 0.04 mg/mL, and the tissue was digested in an incubator at 37 °C, 5% carbon dioxide, for 2 h. After the tissue had been fully ground, the cells were washed and counted to 1×107 cells/mL. Then, 100 μL (1 × 106) cells were added to solution diluted antibody for staining, such as PE anti-mouse CD11b (Biolegend), APC anti-mouse CD206 (Biolegend), FITC anti-mouse F4/80 (Biolegend), and APC anti-mouse Gr-1 (Biolegend), to analyze myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in tumors. APC anti-mouse CD8a (Biolegend), PE-Cy7 anti-mouse CD4 (Biolegend), and PE anti-mouse Ki67 were also used to stain the cells, in order to analyze proliferative T cells in the tumor microenvironment. Flow cytometry was used to wash and analyze the cells, and a Mouse Regulatory T Cell Staining Kit #1 (eBioscience) was used to detect regulatory T cells (Tregs) according to the manufacturer’s instructions.
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