Foxp3 buffer

Manufactured by Thermo Fisher Scientific

The FoxP3 buffer is a specialized buffer solution designed for the intracellular staining and detection of the FoxP3 transcription factor. It is commonly used in flow cytometry applications for the identification and analysis of regulatory T cells (Tregs).

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Lab products found in correlation

Fortessa, by BD (2 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions) Bromodeoxyuridine (brdu), by BD (2 mentions) Brefeldin a, by Merck Group (2 mentions) Fixable viability dye, by Thermo Fisher Scientific (1 mentions) Annexin 5 staining kit, by BD (1 mentions) Lsrii cytometer, by BD (1 mentions) Fluorescently labeled antibodies, by BD (1 mentions) Anti cd16 cd32 clone 2.4g2, by BD (1 mentions) Anti fitc beads, by Miltenyi Biotec (1 mentions) Methanol, by Merck Group (1 mentions) Facsaria 2, by BD (1 mentions) Lsr 2, by BD (1 mentions) Dnase 1, by Roche (1 mentions) Bromodeoxyuridine (brdu), by Roche (1 mentions) Cd44 clone im7, by Thermo Fisher Scientific (1 mentions) Ifnγ clone xmg1, by BioLegend (1 mentions) Mabs to cd8α clone 53 6.7, by Thermo Fisher Scientific (1 mentions) Cd45.1 clone a20, by BioLegend (1 mentions) Tnf α clone tn3 19.12, by Thermo Fisher Scientific (1 mentions)

11 protocols using foxp3 buffer

HBV-specific T cell Responses Evaluation

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Peripheral blood mononuclear cells were stimulated with HBV antigens and genotype-specific peptide pools for 7 days. On day 6, PBMC were subjected to a second round of stimulation with the HBV antigens and the overlapping HBV peptides and stained overnight with CD107a-APC and protein inhibitor cocktail. On day 7, PBMC were surface stained with CD3-BV510, CD4-V450, and CD8-APC-eFluor780 antibodies and stained intracellularly for IFN-γ as described above. PBMC stimulated with PMA/ionomycin were used as a positive control.
For T-regulatory cell staining, PBMC were surface stained with CD3, CD4, and CD25-FITC, fixed and permeabilized with FoxP3 buffer (eBioscience) and stained intracellularly with FoxP3-PerCPCy5.5 antibody as per the manufacturer’s instructions.

Phillips S., Mistry S., Riva A., Cooksley H., Hadzhiolova-Lebeau T., Plavova S., Katzarov K., Simonova M., Zeuzem S., Woffendin C., Chen P.J., Peng C.Y., Chang T.T., Lueth S., De Knegt R., Choi M.S., Wedemeyer H., Dao M., Kim C.W., Chu H.C., Wind-Rotolo M., Williams R., Cooney E, & Chokshi S. (2017). Peg-Interferon Lambda Treatment Induces Robust Innate and Adaptive Immunity in Chronic Hepatitis B Patients. Frontiers in Immunology, 8, 621.

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Immune Cell Phenotyping by Flow Cytometry

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Frozen samples were thawed using RPMI 10% FBS + DNase (15μg/mL). Cells were stained with Cisplatin and DNA as described [17 (link)] After wash, cells were stained in PBS + 0.5% BSA buffer with antibodies at 4 °C for 15mins. After washing twice, cells were fixed in fixation FoxP3 buffer (eBioscience) for 30 min at 4 °C. After washing in perm buffer, cells were stain with αEomes-PE for 30 min at 4 °C in perm buffer. Cells were washed and stained with intracellular markers for 30 min at 4 °C in perm buffer. After washing twice, cells were fixed in PBS 2% PFA overnight.

Xu W., Monaco G., Wong E.H., Tan W.L., Kared H., Simoni Y., Tan S.W., How W.Z., Tan C.T., Lee B.T., Carbajo D., K.G. S., Low I.C., Mok E.W., Foo S., Lum J., Tey H.L., Tan W.P., Poidinger M., Newell E., Ng T.P., Foo R., Akbar A.N., Fülöp T, & Larbi A. (2018). Mapping of γ/δ T cells reveals Vδ2+ T cells resistance to senescence. EBioMedicine, 39, 44-58.

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Multiparametric Flow Cytometry Analysis

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Cells were analyzed on either a BD Fortessa or LSR-II cytometer. SIY-loaded pentamers were from ProImmune. The following antibodies were used in analyses: BD Biosciences: CD45 (30-F11), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), BrdU (Bu20a), Ki-67 (35/Ki-67), and active caspase-3 (C92-605); eBioscience: LAG-3 (C9B7W), 4-1BB (17B5); Biolegend: PD-1 (RMPI-30); Thermo-Fisher: γH2AX (CR55T33). Fixable viability dyes were used to gate out dead cells and were purchased from eBioscience. Tumors, lymph nodes and spleens were dissociated through a 70 µM cell strainer to generate cell suspensions. Tumor suspensions were centrifuged over a Ficoll-hypaque gradient to isolate live mononuclear cells. Cell suspensions were stained with antibodies in PBS containing 1% FBS for 20 min at room temperature. For intracellular antigens, cells were fixed and permeabilized in FoxP3 buffer (eBioscience) for 30 min at room temperature, washed, and stained with intracellular antibodies for 30 min at room temperature. For annexin V staining, cells were first stained with extracellular antibodies and Fixable viability dyes. Immediately before analysis, cells were stained using the annexin V staining kit (BD Biosciences, catalog number 559763).

Horton B.L., Williams J.B., Cabanov A., Spranger S, & Gajewski T.F. (2017). Intratumoral CD8+ T-Cell Apoptosis is a Major Component of T-Cell Dysfunction and Impedes Anti-Tumor Immunity. Cancer immunology research, 6(1), 14-24.

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Peritoneal Mast Cell Characterization

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Peritoneal lavage was performed using 5 mL PBS supplemented with 1% heat-inactivated FBS on healthy female and male mice. Total cell numbers were evaluated by counting cells manually in a Neubauer chamber. Relative percentages of live mast cells were determined by flow cytometry; analysis was performed using FlowJo with single cell gating, dead cell exclusion using propidium iodide (PI) and, mast cells identified as FcεRI⁺, IgE⁺, and, c-kit⁺. Single cell suspensions blocked with anti-CD16/CD32 (clone 2.4G2) and stained for 30–40 min on ice with fluorescently labeled antibodies (BioLegend, BD Biosciences) were acquired using a 4-color FACS Calibur (BD Biosciences). Mast cells were permeabilized using the Foxp3 buffer (eBioscience) and stained using an anti-mouse granzyme A antibody (eBioscience, clone 3G8.5).

Burgener S.S., Brügger M., Leborgne N.G., Sollberger S., Basilico P., Kaufmann T., Bird P.I, & Benarafa C. (2021). Granule Leakage Induces Cell-Intrinsic, Granzyme B-Mediated Apoptosis in Mast Cells. Frontiers in Cell and Developmental Biology, 9, 630166.

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Multiparameter Flow Cytometry Assay for Immune Cell Profiling

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Cells were stained with Fixable Viability Dye eFluor 780 (eBioscience), then stained for surface markers for 25 minutes at room temperature (RT). Cells were then fixed with 2% PFA (EMS)/PBS for 20 minutes at 4°C, or fixed and permeabilized for intracellular/intranuclear staining. For intracellular cytokine staining, cells were permeabilized using BD cytofix/cytoperm, according to the manufacturer’s instructions, then stained in 1x BD fix/perm buffer for 25 minutes at RT. For intranuclear staining, cells were fixed with 4% PFA for 10 minutes at RT, and permeabilized with ice-cold (−20°C) methanol (Sigma) for 15 minutes on ice. Intranuclear staining was then performed in FoxP3 buffer (eBioscience) for 45 minutes at RT. All flow cytometry data was acquired on a BD LSR II or BD Fortessa, and analyses were performed on FlowJo v10.
For cell sorting, freshly isolated PBMC were lineage-depleted with FITC-conjugated antibodies (anti-CD3, CD14, CD19, CD16, CD94, CD11c, CD123) and anti-FITC beads (Miltenyi Biotec) according to the manufacturer’s instructions, with minor modifications. Twenty microliters of beads per 10⁷ cells were used in most experiments. After staining with viability dye and surface markers, cells were sorted on a BD FACS ARIA II using a 70 μm nozzle, and collected into Eppendorf tubes containing cRPMI/10%HS.

Roan F., Stoklasek T.A., Whalen E., Molitor J.A., Bluestone J.A., Buckner J.H, & Ziegler S.F. (2016). CD4+ group 1 innate lymphoid cells form a functionally distinct ILC subset that is increased in systemic sclerosis. Journal of immunology (Baltimore, Md. : 1950), 196(5), 2051-2062.

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BrdU Labeling and Detection Protocol

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BrdU (0.8 mg, BD) was given intraperitoneally 24 h before animal sacrifice. After staining extracellular antigens, cells were fixed and permeabilized with FoxP3 buffer (eBioscience) for 30 min at room temperature. Cells were resuspended in PBS with DNAse I (300 µg/mL, Roche) at 37 °C for 1 h. Cells were subsequently stained with anti-BrdU antibody at room temperature for 30 min.

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Multiparametric Analysis of Splenocyte Responses

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Splenocytes were cultured for 5 h in DMEM containing 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin and supplemented with 1ug/ml brefeldin A (Sigma-Aldrich). Cells were exposed to Fixable Viability Dye eFluor 780 (eBioscience), then surface stained with mAbs to CD8α (clone 53–6.7; eBioscience), CD44 (clone IM7; eBioscience), and CD45.1 (clone A20; BioLegend), permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) or FoxP3 buffer (eBioscience), and stained for intracellular IFNγ (clone XMG1.2; Biolegend), TNFα (clone TN3-19.12; eBioscience), IL-2 (clone JES6-5H4; BD Biosciences), and Nur77 (clone 12.14; eBioscience). D^bLT359 and SiteV tetramers [25 (link), 75 (link)] were provided by the NIH Tetramer Core Facility (Atlanta, GA).

Maru S., Jin G., Schell T.D, & Lukacher A.E. (2017). TCR stimulation strength is inversely associated with establishment of functional brain-resident memory CD8 T cells during persistent viral infection. PLoS Pathogens, 13(4), e1006318.

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Intracellular Antibody Staining in Transfected HeLa Cells

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We transfected HeLa cells using Lipofectamine LTX (Life Technologies) with PR8 proteins HA, NA, M, NP, or NS1 in a pDZ vector and cultured for 24 hr. To enable Ig or VLRB access to internal proteins, we fixed and permeabilized NP, NS1, and M transfected cells with FoxP3 buffer (eBiosciences). We stained all cells with appropriate dye-labeled Ig, mouse sera, or lamprey plasma and analyzed samples using a LSR II flow cytometer and fitted data to a one-site binding hyperbola model using PRISM software.

Altman M.O., Bennink J.R., Yewdell J.W, & Herrin B.R. (2015). Lamprey VLRB response to influenza virus supports universal rules of immunogenicity and antigenicity. eLife, 4, e07467.

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Cytokine-Activated NK Cell Cytotoxicity Assay

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Cells from LN cell suspensions were stimulated overnight in medium containing IL-2 (10 ng/mL) and IL-12 (5 ng/mL) (Miltenyi Biotec) and then co-cultured for 5 h with K562 or MCF7 cells (breast cancer cell line) at a 1:1 effector:target ratio. Co-cultures were performed in U-bottom plates in the presence of Golgi Stop (BD Biosciences), Brefeldin A (BD), and CD107-FITC antibody (Table S2). Cells were then stained with Flexible Viability Dye eFluor 506 (eBioscience) for 30 min before labeling with surface antibodies (Table S2). To stain intracellular IFNγ and TNFα, cells were fixed and permeabilized for 30 min in FoxP3 buffer (eBioscience) before incubating with anti-IFNγ-APC (BD) and anti-TNFα-PECy7 in perm buffer for 30 min.

Rethacker L., Boy M., Bisio V., Roussin F., Denizeau J., Vincent-Salomon A., Borcoman E., Sedlik C., Piaggio E., Toubert A., Dulphy N, & Caignard A. (2022). Innate lymphoid cells: NK and cytotoxic ILC3 subsets infiltrate metastatic breast cancer lymph nodes. Oncoimmunology, 11(1), 2057396.

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Multicolor Flow Cytometry Analysis

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All flow cytometry and fluorescent microscopy antibodies were purchased from ebioscience unless otherwise indicated. CD11c, CD40, CD44, CD80, CD86, CD69, CD25, CD11b, Gr1, Ly6C, CD19, CD138, CD4 (BD Bioscience), and CD8 (BD Bioscience) were used for extracellular staining. BRDU (BD Biosciences) IFNγ, TNFα, IL-12, IL-10, and Foxp3 were used for intracellular staining and Foxp3 Buffer was used for permeabilization (eBioscience).

Thaxton J.E., Liu B., Zheng P., Liu Y, & Li Z. (2014). Deletion of CD24 impairs development of heat shock protein gp96 -driven autoimmune disease through expansion of myeloid derived suppressor cells. Journal of immunology (Baltimore, Md. : 1950), 192(12), 5679-5686.

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