Mouse treg staining kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Mouse Treg Staining Kit is a laboratory tool designed to identify and analyze regulatory T cells (Tregs) in mouse samples. The kit provides the necessary reagents and protocols to detect Tregs through the expression of specific surface markers. It is intended for research use only and does not make any claims about its suitability for diagnostic or therapeutic purposes.

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11 protocols using mouse treg staining kit

Quantitative BALF Cellular Analysis in Mice

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Additional female mice were used to analyze total and differential cell counts in BALF (n = 10/group). For this purpose, a polyethylene cannula was inserted into the trachea and a total volume of 1.0 mL of PBS containing 10 mM EDTA was instilled and aspirated (only once). BALF was centrifuged at 300 × g for 10 minutes at 4 °C. The supernatant was removed and the pellet resuspended in 250 μL PBS. Total leukocyte counts in BALF were quantitated in Neubauer chambers under light microscopy after dilution of the samples in Türk solution (2% acetic acid).
Cell suspensions from BALF were stained with monoclonal anti-mouse CD3 (Pe-Cy5-labeled) and CD4 (FITC-labeled) antibodies (eBioscience, San Diego, CA, USA) to assess CD3⁺CD4⁺ T-cell percentages. The regulatory T cell (Treg) population was characterized by staining for CD4, CD25, and Foxp3, as per manufacturer instructions (Mouse Treg Staining Kit, eBioscience, San Diego, CA, USA). Additionally, anti-mouse Siglec-F antibody (PE-labeled; BD Pharmingen, San Diego, CA, USA) was used to quantify eosinophils in the polymorphonuclear-gated populations in BALF samples. All data were acquired in a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and analyzed using FlowJo X 10.0.7 software (Tree Star Inc., Ashland, OR, USA).

de Castro L.L., Xisto D.G., Kitoko J.Z., Cruz F.F., Olsen P.C., Redondo P.A., Ferreira T.P., Weiss D.J., Martins M.A., Morales M.M, & Rocco P.R. (2017). Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma. Stem Cell Research & Therapy, 8, 151.

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Treg Immunophenotyping and Proliferation

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Lymphocytes were labeled with conjugated mAb in the presence of purified anti-CD16/32 at saturation to block unspecific staining for 30 min at 4 °C. For intracellular staining, lymphocytes were stimulated for 4 hours with phorbol-12-myristate-13-acetate (50ng/mL) and ionomycin (500ng/mL), with brefeldin A added for the final 2 hours. Tregs were detected by a mouse Treg staining kit (eBioscience, San Diego, CA). In vivo proliferation was assessed with a BD Pharmingen BrdU APC kit. Cells were analyzed on a FACS Calibur flow cytometer with CellQuest (BD Bioscience, San Diego, CA).

Liu Y., Wu Y., Wang Y., Cai Y., Hu B., Bao G., Fang H., Zhao L., Ma S., Cheng Q., Song Y., Liu Y., Zhu Z., Chang H., Yu X., Sun A., Zhang Y., Vignali D.A., Wu D, & Liu H. (2014). IL-35 mitigates murine acute graft-versus-host disease with retention of graft-versus-leukemia effects. Leukemia, 29(4), 939-946.

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Treg Immunophenotyping and Proliferation

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Regulatory T Cell Profiling in Murine Tissues

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Single cell suspensions from skin, tumor, draining lymph nodes (dLN) and spleen were stained with fluorochrome-conjugated antibodies against CD11b (PA5-79532, ThermoFisher), CD4 (116005, BioLegend), CD8 (100726, BioLegend), CD25 (102011, BioLegend), Foxp3 (320105, BioLegend), Ly6C (128021, BioLegend) or Ly6G (127607, BioLegend) and appropriate isotype controls (BioLegend) for 30 min on ice. Cells were washed and fixed with 1% paraformaldehyde and analyzed on a FACSAriaIIu (BD Biosciences). The frequency of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) was determined using the mouse Treg staining kit (eBioscience).

Pinto N.A., Abba M.C., Laporte L., Pérez Sáez J.M., Blidner A.G., Torres N.I., Morales R.M., Gatto S.G., Bach C.A., Veigas F., García Rivello H.J., Song P., Frederiksen J.H., Rasmussen L.J., Poirier F., Croci D.O., Sundblad V., Rabinovich G.A, & Cerliani J.P. (2023). Galectin-7 reprograms skin carcinogenesis by fostering innate immune evasive programs. Cell Death and Differentiation, 30(4), 906-921.

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Measuring Mouse Splenic Treg Cells

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To measure the percentage of Tregs, a single-cell suspension of splenic cells was prepared according to a previously described method (12 (link)). The cells were stained with a mouse Treg staining kit (eBioscience) and analyzed with Cell Quest software. Cell suspensions were stained by adding 1 μL of FITC-labeled anti-mouse CD4, 1 μL of PE-labeled anti-CD25 mAb, and 2 μL of APC-labeled anti-mouse Foxp3 (clone PC61), and APC-conjugated rat IgG2α served as isotype control. Then sorted for the three target populations by flow cytometry using a FACSCalibur system (Becton Dickinson).

Tang C.L., Yu X.H., Li Y., Zhang R.H., Xie J, & Liu Z.M. (2019). Schistosoma japonicum Soluble Egg Antigen Protects Against Type 2 Diabetes in Leprdb/db Mice by Enhancing Regulatory T Cells and Th2 Cytokines. Frontiers in Immunology, 10, 1471.

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Flow Cytometry Analysis of IL-10+ B Regs

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For flow cytometry analysis of IL-10-producing CD19⁺ B_regs, cells in splenocytes were analyzed for the expression of surface markers using the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD19 (clone 1D3; BD Biosciences, San Jose, CA, USA) and phycoerythrin (PE)-conjugated anti-IL-10 (clone JES5-16E3; eBioscience, San Diego, CA, USA). For subsequent IL-10intracellular staining, the Cytofix/Cytoperm kit (BD Biosciences) was used according to the manufacturer's protocol (BD Biosciences). For the analysis of CD4⁺CD25⁺FoxP3⁺ T_reg cells, the mouse T_reg-staining kit was used according to the manufacturer's instructions (eBioscience). The stained cells were analyzed by 3-5 color flow cytometry using a FACSversa flow cytometer (BD Biosciences). The FlowJo software (Tree Star Inc., OR, USA) was used to analyze flow cytometry data. To determine background staining, non-reactive iso-type-matched control monoclonal antibodies (eBioscience) were used and gated to exclude ≥98% of non-reactive cells.

Jeong Y.I., Hong S.H., Cho S.H., Lee W.J, & Lee S.E. (2015). Toxoplasma gondii Infection Suppresses House Dust Mite Extract-Induced Atopic Dermatitis in NC/Nga Mice. Allergy, Asthma & Immunology Research, 7(6), 557-564.

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Cell Cycle and Treg Analysis of MSCs

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For cell cycle analysis, MSCs collected and fixed with 70% ethanol at -20°C for 24 h. After being washed with PBS and then treated with 100 μg/ml RNase (Sigma, USA) for 20 min, cells stained with 50 μg/ml propidium iodide (PI) solution (Sigma, USA) for 20 minutes and analyzed by the flow cytometry machine (FACS Calibur, BD Biosciences, USA). The fraction of cells in the G0/G1, S, and G2/M phases were quantified with the ModFit LT system.
For Treg cell analysis, Cells obtained from lymph nodes, spleen and peripheral blood were blocked with anti-CD16/32 and stained with monoclonal anti-mouse CD4 antibody (eBiosciences; San Diego, CA, USA) to assess T cell percentages. Treg cells were characterized by staining for CD4, CD25 and Foxp3, according to the manufacturer’s instructions (Mouse Treg Staining Kit, eBiosciences). All data were acquired in a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and analysed using FlowJo X 10.0.7 software.

Ji J., Fu T., Dong C., Zhu W., Yang J., Kong X., Zhang Z., Bao Y., Zhao R., Ge X., Sha X., Lu Z., Li J, & Gu Z. (2019). Targeting HMGB1 by ethyl pyruvate ameliorates systemic lupus erythematosus and reverses the senescent phenotype of bone marrow-mesenchymal stem cells. Aging (Albany NY), 11(13), 4338-4353.

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Quantification of Regulatory T Cells

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To evaluate regulatory the Treg percentage, single-cell suspensions of splenocytes were prepared according to the method described by Mo et al. (19 (link)). Briefly, resected spleens were ground and filtered through a 200-mesh filter cloth. Red blood cells in the generated cell pellet were cleaved using an erythrocyte lysate (Beyotime, China), and 5 × 10⁶ cells were suspended in Roswell Park Memorial Institute (RPMI)-1640 medium. The cells were then stained with conjugated antibodies including FITC-conjugated anti-mouse CD4, APC-conjugated anti-mouse CD25, and PE-conjugated anti-mouse Foxp3, using a mouse Treg-staining Kit (eBioscience). Finally, they were analyzed using a BD Biosciences FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA).

Tang C.L., Pan Q., Xie Y.P., Xiong Y., Zhang R.H, & Huang J. (2019). Effect of Cytotoxic T-Lymphocyte Antigen-4 on the Efficacy of the Fatty Acid-Binding Protein Vaccine Against Schistosoma japonicum. Frontiers in Immunology, 10, 1022.

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Comprehensive Immune Cell Profiling

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Cells were blocked with normal sheep serum and stained with monoclonal anti-mouse CD4 antibodies (RM4-5 clone; eBiosciences; San Diego, CA, USA). Intracellular staining of CD4 T cells was performed using anti-mouse IL-10-PE antibody (JES5-16E3 clone), anti-IL-4 PECy7 (11B11 clone), and anti-IL-17 eFluor 660 (eBio17B7 clone) or the respective controls IgG2bκ PE, IgG1κ PECy7, and IgG2aκ eFluor 660 (all against mouse proteins, from eBioscience). Treg cells were characterized by staining for CD4, CD25, and Foxp3, according to the manufacturer's instructions (Mouse Treg Staining Kit, eBiosciences).
Pulmonary myeloid dendritic cells (CD11c⁺CD11b⁺Gr1⁻B220⁻) were characterized as previously [26 (link)], using anti-Gr1 FITC (RB6-8C5 clone) anti-CD11c PE (N418 clone), anti-CD11b PEcy7 (M1/70 clone), and anti-B220 APC (RA3-6B2 clone) or the respective controls IgG2bκ FITC, IgG armenian hamster PE, IgG2bκ PECy7, and IgG2aκ APC (all against mouse proteins, from eBiosciences).
All data were acquired in a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and analyzed using the FlowJo X 10 and 7.6.5 software (Tree Star Inc., Ashland, OR, USA). Percentages of total cells were presented in bar graphs.

Azevedo C.T., Cotias A.C., Arantes A.C., Ferreira T.P., Martins M.A, & Olsen P.C. (2021). Assessment of Allergen-Responsive Regulatory T Cells in Experimental Asthma Induced in Different Mouse Strains. Mediators of Inflammation, 2021, 7584483.

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Treg Cell Analysis by FACS

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Cell culture procedure was the same as for non-specific T cell proliferation assay. Tregs were analyzed by FACS using a mouse Treg staining kit (affymetrix eBioscience).

Pu S., Qin B., He H., Zhan J., Wu Q., Zhang X., Yang L., Qu C, & Zhou Z. (2016). Identification of early myeloid progenitors as immunosuppressive cells. Scientific Reports, 6, 23115.

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