Alexa Fluor 488–conjugated anti-mouse CD45, anti-human CD45, violet 421–conjugated anti-mouse CD4 and CD8, anti-human CD4 and CD8, and phycoerythrin-conjugated anti-mouse or anti-human CD28, 41BB, NKG2D, CD39, PD-1, LAG3, TIM3, and FOXP3, as well as isotype control antibodies, were purchased from Biolegend (San Diego, CA). Rabbit anti-mouse and human CD3 and rabbit anti-human CD8 antibodies were purchased from Abcam (Cambridge, MA). Biotin anti-mouse NKG2D, mouse anti-human NKG2D, rabbit anti-human FOXP3 and anti-human Tbet antibodies were purchased from R&D Systems (Minneapolis, MN). Anti-mouse FOXP3 antibody was purchased from Thermo Fisher. Horseradish peroxidase–conjugated anti-rabbit IgG was purchased from Cell Signaling Technology (Danvers, MA).
Anti mouse foxp3 antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-mouse Foxp3 antibodies are a class of laboratory reagents used in immunological research applications. These antibodies specifically target and bind to the Foxp3 protein, which is a transcription factor expressed in regulatory T cells (Tregs). The core function of these antibodies is to facilitate the identification and analysis of Tregs in mouse models and samples.
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Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Anti nk1, by BioLegend (2 mentions)
Anti tnf α, by BioLegend (2 mentions)
Igg isotype control, by BioLegend (2 mentions)
Anti nk1, by Thermo Fisher Scientific (2 mentions)
Anti il 10, by BioLegend (2 mentions)
Anti il 17a, by BioLegend (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Anti cd4, by Thermo Fisher Scientific (2 mentions)
Anti il 4, by BioLegend (2 mentions)
Anti cd3, by Thermo Fisher Scientific (2 mentions)
Anti cd19, by BioLegend (2 mentions)
Anti cd4, by BioLegend (2 mentions)
Anti ifn γ, by BioLegend (2 mentions)
Anti cd44, by BioLegend (2 mentions)
Anti cd62l, by BioLegend (2 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Rabbit anti human cd8, by Abcam (1 mentions)
Tim 3, by BioLegend (1 mentions)
Ic fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti mouse foxp3 antibody
1
Multiparameter Immune Cell Profiling
2
Isolation and Analysis of Lung and Spleen Immune Cells in Mice
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On day 6 and 31, cells were isolated from the right lung and spleens of mice and incubated with an extracellular antibody mix for inflammatory cell staining (Supplementary Methods 4 and 5 ). For fixation, eBioscience IC Fixation/Permeabilization buffer was used for 30 minutes at room temperature (RT).
For intracellular staining, cells were permeabilized twice using permeabilization buffer at ×1 (Thermo Fisher Scientific) and incubated with anti-mouse Foxp3 antibody (clone FJK-16s), for 1 hour at RT. After incubation, cells were washed with permeabilization buffer, centrifuged at 620g at RT for 2 minutes, and resuspended in fluorescence-activated cell sorting buffer for flow cytometry analysis. Samples were analyzed with a BD LSRFortessa analyzer, and data analysis was performed using FlowJo version 10 software (BD Biosciences) (Supplementary Figure 2 and 3 ).
For intracellular staining, cells were permeabilized twice using permeabilization buffer at ×1 (Thermo Fisher Scientific) and incubated with anti-mouse Foxp3 antibody (clone FJK-16s), for 1 hour at RT. After incubation, cells were washed with permeabilization buffer, centrifuged at 620g at RT for 2 minutes, and resuspended in fluorescence-activated cell sorting buffer for flow cytometry analysis. Samples were analyzed with a BD LSRFortessa analyzer, and data analysis was performed using FlowJo version 10 software (BD Biosciences) (
3
Isolation and Analysis of Tumor-Infiltrating Immune Cells
4
Multiparameter Flow Cytometry Analysis
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Fluorescently labeled antibodies specific for CD3 (Alexa Fluor 700; Life Technologies, Carlsbad, CA, USA), CD4 (PE or Pacific Blue), CD8 (allophycocyanin [APC]), CD19 (peridinin chlorophyll-cyanine 5.5 [PerCP-Cy5.5]), CD25 (PerCP-Cy7), and CD62L (fluorescein isothiocyanate [FITC]) were obtained from BD Biosciences (San Jose, CA, USA). Intracellular staining for Foxp3 was performed using anti-mouse Foxp3 antibody labeled with FITC according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA). Labeled cells were washed with phosphate-buffered saline (PBS), and a minimum of 10,000 cells were analyzed from each sample using a 5-laser (355, 405, 488, 561, and 640 nm) BD LSR II flow cytometer (BD Biosciences). To determine cell numbers, 50 μl of Molecular Probes CountBright beads (Thermo Fisher Scientific, Eugene, OR, USA) were added to each sample before cytometric analyses, and the number of cells per unit volume was calculated as (cell events/bead events) × (bead count/volume of blood sample analyzed). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).
5
Quantification of Murine Myeloid-Derived Suppressor Cells and Regulatory T Cells
6
Liver Leukocyte Isolation and Flow Cytometry
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Leukocytes were isolated from the liver in the way as described previously40 (link). For flow cytometry, cells were stained for surface antigens with anti-NK1.1, anti-CD3, anti-CD4, anti-CD8α, anti-CD19, anti-CD44, anti-CD62L (BioLegend, San Diego, CA), or/and for intracellular antigens with anti-IFN-γ, anti-IL-4, anti-IL-17A, anti-TNF-α and anti-IL-10 (BioLegend). IgG isotype controls (Biolegend) were used in parallel. To detect Treg cells, cells were stained with anti-NK1.1, anti-CD3, anti-CD4, anti-CD25 at 4 °C for 30 minutes; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). Methods were the sameas those described previously9 (link).
7
Flow cytometric analysis of T cell subsets
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Cells surface markers and intracellular cytokines were stained according to the eBioscience flow cytometry protocol. Briefly, for surface staining of CD3, CD4, and CD25, cells were resuspended in PBS containing 0.5% BSA and were incubated on ice for 30 min with fluorochrome-conjugated antibodies (eBioscience). After fixation and permeabilization, cells were stained, respectively, with anti-mouse IL-17A, anti-mouse IFN-γ, and anti-mouse Foxp3 antibodies conjugated with fluorochrome (eBioscience). Finally, stained cells were fixed with 4% paraformaldehyde and analyzed on a BD Caliber flow cytometer.
8
Flow Cytometry Analysis of Th17 and Treg Cells
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Flow cytometry was performed as described elsewhere [28 (link)–29 (link)]. In brief, for Th17 detection, approximately 106 splenocytes were prepared and incubated with PMA (20 ng/m) and ionomycin (1 μg/mL) in the presence of monensin (2 mmol/ml, Sigma-Aldrich, St. Louis, MO, USA) for 4 h (37°C, 5% CO2). The cells were subjected to allophycocyanin (APC)-anti-CD3 and phycoreythrin (PE)-Cy5-anti-CD4 staining at 4°C for 30 minutes in the dark. After surface staining, the cells were fixed in paraformaldehyde and permeabilized in Perm/Fix solution according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA) and then intracellularly stained with anti-mouse PE-IL-17. To detect Treg cells, the cell suspensions were stained with APC-anti-CD3, PE-cy5-anti-CD4, and fluorescein isothiocyanate (FITC)-anti-CD25 at 4°C for 30 minutes; treated with eBioscience Perm/Fix mixture; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). All of the stained cells were evaluated using a flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed using Diva software (BD Biosciences).
9
Liver Leukocyte Isolation and Flow Cytometric Analysis
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Leukocytes were isolated from liver as described previously52 (link). For flow cytometry, cell preparations were incubated with purified anti-CD16/CD32 antibody (BioLegend, San Diego, CA) for 15 min at 4 °C, and then stained with the following antibodies conjugated to fluorescent labels: anti-NK1.1, anti-CD3, anti-CD4, anti-CD8β, anti-CD19, anti-CD44, anti-CD62L, LAG3 and 2B4 (BioLegend).
For intracellular cytokine staining, approximately 106 liver mononuclear cells were stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience, California, USA) for 4 h, or EmP (5 μg/ml) for 10 h (37 °C, 5% CO2) as previously described52 (link). The cells were stained for surface anti-NK1.1, anti-CD3, anti-CD4, anti-CD8β, LAG3 and 2B4, and then stained for intracellular anti-IFN-γ, anti-IL-4, anti-IL-17A, anti-TNF-α, anti-IL-10 and granzyme B (Biolegend). IgG isotype controls (Biolegend) were used in parallel. To detect Treg cells, cells were stained with anti-NK1.1, anti-CD3, anti-CD4, anti-CD25 at 4 °C for 30 minutes; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). ALSRFortessa flow cytometry (BD Immunocytometry Systems, San Jose, CA) was used to acquire data, which were analyzed with Flowjo software (Treestar, Inc., Ashland, OR).
For intracellular cytokine staining, approximately 106 liver mononuclear cells were stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience, California, USA) for 4 h, or EmP (5 μg/ml) for 10 h (37 °C, 5% CO2) as previously described52 (link). The cells were stained for surface anti-NK1.1, anti-CD3, anti-CD4, anti-CD8β, LAG3 and 2B4, and then stained for intracellular anti-IFN-γ, anti-IL-4, anti-IL-17A, anti-TNF-α, anti-IL-10 and granzyme B (Biolegend). IgG isotype controls (Biolegend) were used in parallel. To detect Treg cells, cells were stained with anti-NK1.1, anti-CD3, anti-CD4, anti-CD25 at 4 °C for 30 minutes; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). ALSRFortessa flow cytometry (BD Immunocytometry Systems, San Jose, CA) was used to acquire data, which were analyzed with Flowjo software (Treestar, Inc., Ashland, OR).
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