Mouse th17 treg phenotyping kit

Manufactured by BD

Sourced in United States

The Mouse Th17/Treg Phenotyping Kit is a laboratory tool designed to analyze the frequencies of Th17 and regulatory T (Treg) cells in mouse samples. The kit provides the necessary reagents and protocols to identify and quantify these cell populations through flow cytometry.

Automatically generated - may contain errors

Lab products found in correlation

9 protocols using mouse th17 treg phenotyping kit

Phenotyping Th17 and Treg Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

The lymph nodes (pool of inguinal and mesenteric) were collected on the 7^th day after immunization. The cells were isolated by flushing the tissue through a cell strainer (70 μm) with RPMI medium, followed by centrifugation at 450 g for 5 min at 4 °C and the pellet was resuspended in 3 ml of RPMI medium. In a 24-well plate, the cells were cultured at a concentration of 1 × 10⁶ cells in 500 μl in RPMI medium with or without MOG_35–55 (1 µg/ml) and BD GolgiStop™ Protein Transport Inhibitor (provided in the kit or Cat #554724) for 12 h. After 8 h, cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 4 h. The cells were stained for surface markers and intracellular cytokine with Mouse Th17/Treg Phenotyping kit (#560767, BD Biosciences) according to the manufacturer’s directions. Samples were analyzed using a FACS Canto II Flow Cytometer (BD Biosciences, San Diego, USA) after the acquisition. Data analysis was performed using FlowJo VX.0.7r2 (Tree Star, Ashland, OR). The strategy for analysis and representative dot plots of the experiments are shown in the Supplementary Material.

Sant'Anna M.B., Giardini A.C., Ribeiro M.A., Lopes F.S., Teixeira N.B., Kimura L.F., Bufalo M.C., Ribeiro O.G., Borrego A., Cabrera W.H., Ferreira J.C., Zambelli V.O., Sant'Anna O.A, & Picolo G. (2020). The Crotoxin:SBA-15 Complex Down-Regulates the Incidence and Intensity of Experimental Autoimmune Encephalomyelitis Through Peripheral and Central Actions. Frontiers in Immunology, 11, 591563.

+ Open protocol

+ Expand

Immunophenotyping of Murine Lymphocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols

The spleen was removed from the mice and dissected. Splenic cell suspensions were gently pressed through a sterile 100 μm nylon mesh, and lymphocytes were isolated by ficoll gradient centrifugation (GE Healthcare, Sweden). The isolated lymphocytes were stained with a mouse Th1/Th2/Th17 phenotyping kit (the fluorescent antibodies: CD4, IFN-γ, IL-4 or IL-17A for detecting Th1, Th2 or Th17) and a mouse Th17/Treg phenotyping kit (the fluorescent antibodies: Foxp3 and CD4 for detecting Tregs) (BD Pharmingen, USA), and were acquired in a BD LSR II flow cytometer (BD Biosceinces, USA).

Rau C.S., Lin M.W., Wu S.C., Wu Y.C., Lu T.H., Tzeng S.L., Chen Y.C., Wu C.J, & Hsieh C.H. (2015). Regulatory and Effector Helper T-Cell Profile after Nerve Xenografting in the Toll-Like Receptor-Deficient Mice. International Journal of Medical Sciences, 12(8), 650-654.

+ Open protocol

+ Expand

Splenocyte Th17/Treg Phenotyping

Check if the same lab product or an alternative is used in the 5 most similar protocols

The differentiated splenocytes were fixed with fixation buffer and stained with mouse Th17/Treg phenotyping kit (BD Pharmingen™) containing antibodies against mouse CD4 conjugated with peridinin-chlorophyll-protein (PerCP)-Cy5.5, mouse IL-17A conjugated with phycoerythrin (PE), and mouse Foxp3 conjugated with Alexa Fluor^®647. The staining procedures were performed according to the manufacturer’s instructions. Approximately 10,000 cells were analyzed using BD FACSCANTO II flow cytometry (BD Biosciences). The control cells were stained with isotype antibodies conjugated with fluorophores and served as the control staining group to set up the gating. Th17 population was defined as CD4⁺IL-17A⁺ and Treg population was defined as CD4⁺Foxp3⁺.

Chen R.Y., Fan Y.M., Zhang Q., Liu S., Li Q., Ke G.L., Li C, & You Z. (2015). Estradiol Inhibits Th17 Cell Differentiation through Inhibition of RORγT Transcription by Recruiting ERα/REA Complex to the EREs of RORγT Promoter. Journal of immunology (Baltimore, Md. : 1950), 194(8), 4019-4028.

+ Open protocol

+ Expand

Multiparametric Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

LPLs were isolated and stimulated with Phorbol 12-myristate 13-acetate and ionomycin in the presence of brefeldin and monensin for 4 h at 37°C in a humidified CO₂ incubator. Stimulated cells were stained for surface antigens with fluorescently labelled antibodies. Cells were then stained with fixable viability dye FV510 for live/dead discrimination followed by intracellular staining using Fix/Perm transcription factor kit (BD Biosciences, Franklin Lakes, NJ) and fluorescently labeled antibodies as follows: IL4 - PE-Cy7 (560699; BD Pharmingen, Franklin Lakes, NJ), KLRG1 – PerCP-Cy5.5 (563595; BD Pharmingen), IL22 – PE (BD Pharmingen; 516404), IFNγ- APC-Cy7 (561479; BD Pharmingen), Ly6G- FITC (BD Pharmingen; 561105), NK1.1 -FITC (561082; BD Pharmingen), CD3- FITC (561798; BD Pharmingen), CD19- FITC (BD Pharmingen; 561740), B220- FITC (561877; BD Pharmingen), NKp46- AF700 (561169; BD Pharmingen), RORγt- AF647 (562682; BD Pharmingen), viability dye FV510- BV510 (BD Pharmingen; 564406), CD127- BV421 (566377; BD Pharmingen), and CD45- BV786 (565477; BD Pharmingen). For Tregs quantification, Mouse Th17/Treg Phenotyping Kit (BD Biosciences) was used. Data were acquired on BD FACSCanto flow cytometer and analyzed using FlowJo software (FlowJo, Ashland OR).

Leon-Coria A., Kumar M., Workentine M., Moreau F., Surette M, & Chadee K. (2020). Muc2 Mucin and Nonmucin Microbiota Confer Distinct Innate Host Defense in Disease Susceptibility and Colonic Injury. Cellular and Molecular Gastroenterology and Hepatology, 11(1), 77-98.

+ Open protocol

+ Expand

Detecting Splenic Treg Populations

Check if the same lab product or an alternative is used in the 5 most similar protocols

The Tregs (CD4⁺FoxP3⁺) populations in the spleens were detected by using Mouse Th17/Treg Phenotyping Kit (BD Pharmingen) according tothe manufacture instructions.

Li H., Jiang Y.M., Sun Y.F., Li P., Dang R.J., Ning H.M., Li Y.H., Zhang Y.J., Jiang X.X., Guo X.M., Wen N., Han Y., Mao N., Chen H, & Zhang Y. (2014). CCR7 Expressing Mesenchymal Stem Cells Potently Inhibit Graft-versus-Host Disease by Spoiling the Fourth Supplemental Billingham’s Tenet. PLoS ONE, 9(12), e115720.

+ Open protocol

+ Expand

Extraction and Analysis of Murine Lung Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

At the experimental endpoint, K‐RAS‐mutated tumor harboring lungs were harvested, and the lung tissue was mechanically and enzymatically disrupted for cell isolation. Briefly, lungs were put into isolation medium (RPMI with 5% FBS) and minced with scissors. Minced pieces were then transferred into a falcon tube containing isolation medium supplemented with DNase I (50 U/ml, Sigma, St.Louis, MO) and Collagenase I (Gibco, 150 U/ml), and incubated for 1 hr at 37°C. Subsequently, the cell suspension was filtered through 70 and 40 μm cell strainers. For subsequent flow cytometric analysis, cells were stained with the antibodies indicated in Table S5 using a standard protocol. For identification of Th17 and regulatory T cells, the mouse Th17/Treg phenotyping kit (#560767, BD Pharmingen, Franklin Lakes, NJ) was used. All cells are CD45⁺. Stained samples were measured using the BD FACSCanto™ II system. For data analysis, the FlowJo software (TreeStar, Inc., Ashland, OR) was used.

Mohrherr J., Haber M., Breitenecker K., Aigner P., Moritsch S., Voronin V., Eferl R., Moriggl R., Stoiber D., Győrffy B., Brcic L., László V., Döme B., Moldvay J., Dezső K., Bilban M., Popper H., Moll H.P, & Casanova E. (2019). JAK–STAT inhibition impairs K‐RAS‐driven lung adenocarcinoma progression. International Journal of Cancer, 145(12), 3376-3388.

+ Open protocol

+ Expand

Comprehensive Tumor Immune Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols

To analyze the cells of the innate and adaptive immunity associated with cancer progression such as TAMs, TILs, Tregs, Th17 lymphocytes, CTLs, and NK cells, defrosted cells obtained from the lungs and subcutaneously growing tumors were assayed in the FACS Calibur flow cytometer (Becton Dickinson, Warsaw, Poland) using the following monoclonal antibodies: FITC Rat Anti-Mouse CD45, APC Rat Anti-CD11b, PE Rat Anti-Mouse Ly-6G and Ly-6C, PerCP-Cy™5.5 Hamster Anti-Mouse CD3e, PerCP-Cy™5.5 Rat Anti-Mouse CD8a, PE Hamster Anti-Mouse CD178, FITC Rat Anti-Mouse CD49b, Alexa Fluor^® 647 Rat Anti-Mouse CD335 (NKp46), and Mouse Th17/Treg Phenotyping Kit (all from BD Pharmingen), BB515 Rat Anti-Mouse CD25 (BD Horizon).
After defrosting, the obtained serum samples, after defrosting, were assayed for the levels of: IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α, using the Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit (BD).
All determinations were performed in triplicates. The obtained data were analyzed with the use of the CellQuest software (Becton-Dickinson) and FCAP Array software (Becton-Dickinson).

Nowosielska E.M., Cheda A., Pociegiel M., Cheda L., Szymański P, & Wiedlocha A. (2021). Effects of a Unique Combination of the Whole-Body Low Dose Radiotherapy with Inactivation of Two Immune Checkpoints and/or a Heat Shock Protein on the Transplantable Lung Cancer in Mice. International Journal of Molecular Sciences, 22(12), 6309.

+ Open protocol

+ Expand

Murine Th17 and Treg Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Bronchoalveolar lavage fluid extracted from mice was digested with enzymes, and undigested sections were filtered using strainers. Then, Mouse Th17/Treg phenotyping kit (560767; BD Biosciences, USA) was employed to stain Th17 and Treg cells according to the manufacturer's instructions. A flow cytometer (CytoFLEX; Beckman, USA) was used to record the number of specific cells (1×10 4 cells were tested).

Shen M., Lin B., Qian F., Zhao L., Xi Y, & Qian Y. (2022). Taxifolin ameliorates sepsis-induced lung capillary leak through inhibiting the JAK/STAT3 pathway. Allergologia et immunopathologia, 50(2).

+ Open protocol

+ Expand

Quantification of Circulating Monocytes and Lymphocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols

Circulating monocytes were determined in 10 ml of heparinised whole blood incubated for 30 min at RT with Ly6C-PerCP (BD Pharmingen, Madrid, Spain) and CD115-APC (Biolegend, San Diego, CA, USA). For lymphocytes, 10 ml of heparinised whole blood was incubated for 30 min RT with 5 ml Brilliant Stain Buffer (563794, BD) and with Brilliant violet (BV)-rat anti-mouse CD4 (562891, BD), BV-rat anti-mouse CD8a (563068, BD), PE-hamster anti-mouse CD69 (553237, BD) and APC-hamster antimouse CD3e (553066, BD). Incubation with lysing solution (BD Facs Lysing solution) was done before analysis by flow cytometry (FACSVerse BD Biosciences). To detect functionally-polirised CD4CT lymphocyte, 100 ml of heparinised whole blood was stained with the mouse Th17/Treg phenotyping kit (BD Pharmingen, Madrid, Spain) to detect CD4CFoxp3C and CD4CIL17 cells. Analysis of Ly6C low -and Ly6C hi -subsets were determined in CD115C populations.

Andrés-Blasco I., Herrero-Cervera A., Vinué Á., Martínez-Hervás S., Piqueras L., Sanz M.J., Burks D.J, & González-Navarro H. (2015). Hepatic lipase deficiency produces glucose intolerance, inflammation and hepatic steatosis. The Journal of endocrinology, 227(3).

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!