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Foxp3 transcription factor staining buffer set

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The Foxp3/Transcription Factor Staining Buffer Set is a laboratory reagent used for the intracellular staining and detection of Foxp3, a transcription factor. The set includes the necessary buffers and solutions to facilitate the fixation, permeabilization, and staining of cells for the analysis of Foxp3 expression by flow cytometry or other techniques.

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Lab products found in correlation

Fortessa flow cytometer, by BD (2 mentions) Phosflow perm buffer 3, by BD (2 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions) Nab jes6 1a12, by BioXCell (2 mentions) Basiliximab, by Absolute Antibody (2 mentions) Daclizumab, by Absolute Antibody (2 mentions) Zombie aqua viability dye, by BioLegend (2 mentions) Aurora flow cytometer, by Cytek (2 mentions) Lsrfortessa flow cytometer, by BD (2 mentions) Cd25 apc, by Thermo Fisher Scientific (1 mentions) Foxp3 apc, by Thermo Fisher Scientific (1 mentions) Foxp3 pe, by Thermo Fisher Scientific (1 mentions) Cd25 pe, by BioLegend (1 mentions) Lsrii cytometer, by BD (1 mentions) Cd4 fluorescein isothiocyanate, by Thermo Fisher Scientific (1 mentions) Cd8a pe cyanine7, by Thermo Fisher Scientific (1 mentions) Accuri c6 flow cytometer, by BD (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Facsaria cell sorter, by BD (1 mentions) Lsrii analyzer, by BD (1 mentions)

Close spelling variants of this product

Transcription Factor Buffer Set (206 citations) Staining buffer (101 citations) FoxP3 Buffer Set (16 citations) Foxp3 Staining Buffer Set (10 citations) Transcription Factor Buffer (9 citations) Transcription Buffer Set (8 citations) Transcription Factor Staining Buffer Set (5 citations) Foxp3 Transcription Factor Staining Buffer (3 citations) Foxp3/Transcription Factor Buffer Set (2 citations) FoxP3 staining buffer (2 citations)

15 protocols using foxp3 transcription factor staining buffer set

1

Phenotypic analysis of lymphocyte subsets

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The cells were incubated with anti‐CD16/CD32 (KT1632, Invitrogen, Thermo Fisher Scientific) for 15 minutes at 4 ℃. Single‐cell suspensions of splenocytes and peripheral blood mononuclear cells were stained with CD4 fluorescein isothiocyanate (100510, BioLegend, San Diego, CA, USA) and CD25 PE (102007, BioLegend). Mononuclear cells isolated from the brains were stained with CD3e PerCP‐Cyanine5.5 (45‐0031‐82, eBioscience, San Diego, CA, USA), CD4 fluorescein isothiocyanate (11‐0041‐82, eBioscience, ), CD8a PE‐Cyanine7 (25‐0081‐82, eBioscience), and CD25 APC (17‐0257‐42, eBioscience) for 30 minutes at 4℃. Intracellular staining with Foxp3 APC (17‐5773‐82, eBioscience) for splenocytes and peripheral blood mononuclear cells and Foxp3 PE (12‐4777‐42, eBioscience) for mononuclear cells isolated from brains was followed by incubation for 30 minutes at 4℃ after cell fixation and permeabilization using the Foxp3/Transcription Factor Staining Buffer Set (562574, BD Pharmingen Inc., CA, USA) according to the manufacturer’s instructions. Finally, data were acquired with an LSRII cytometer (BD Biosciences, San Jose, CA, USA) and analysis was performed using FlowJo (TreeStar, Ashland, OR, USA).
Yu H., Ma X., Ma X., Chen M., Chu Y., Wu L., Wang W., Qin C, & Tian D. (2021). Remote Limb Ischemic Postconditioning Protects Against Ischemic Stroke by Promoting Regulatory T Cells Thriving. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 10(22), e023077.
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2

Intracellular Signaling Cytometry Assay

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Cells were stimulated for 5h with PMA (200 nM) and ionomycin (1 μg/ml). For the last 2.5h of stimulation, brefeldin A (10 μg/ml) was added to the cells. Cells were fixed with paraformaldehyde (PFA, 4%) and permeabilized with 0.5% BSA and saponine. The antibodies used are listed in Suppl. Table 9. For NF-κB p65 phosphorylation, cells were stimulated for 5-30 min with PMA and ionomycin, fixed with PFA and permeabilized with methanol. For intracellular FOXP3 staining, a FOXP3 Transcription Factor Staining Buffer Set (BD Bioscience) was used. All samples were acquired on a Fortessa flow cytometer (BD Bioscience) and data was analyzed with FlowJo Software.
Emming S., Bianchi N., Polletti S., Balestrieri C., Leoni C., Montagner S., Chirichella M., Delaleu N., Natoli G, & Monticelli S. (2020). A molecular network regulating the pro-inflammatory phenotype of human memory T lymphocytes. Nature immunology, 21(4), 388-399.
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3

Intracellular Signaling Cytometry Assay

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Cells were stimulated for 5h with PMA (200 nM) and ionomycin (1 μg/ml). For the last 2.5h of stimulation, brefeldin A (10 μg/ml) was added to the cells. Cells were fixed with paraformaldehyde (PFA, 4%) and permeabilized with 0.5% BSA and saponine. The antibodies used are listed in Suppl. Table 9. For NF-κB p65 phosphorylation, cells were stimulated for 5-30 min with PMA and ionomycin, fixed with PFA and permeabilized with methanol. For intracellular FOXP3 staining, a FOXP3 Transcription Factor Staining Buffer Set (BD Bioscience) was used. All samples were acquired on a Fortessa flow cytometer (BD Bioscience) and data was analyzed with FlowJo Software.
Emming S., Bianchi N., Polletti S., Balestrieri C., Leoni C., Montagner S., Chirichella M., Delaleu N., Natoli G, & Monticelli S. (2020). A molecular network regulating the pro-inflammatory phenotype of human memory T lymphocytes. Nature immunology, 21(4), 388-399.
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4

Multicolor Flow Cytometry Analysis

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Cell surface, intracellular cytokine and Foxp3 were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, PE combined with a cyanine dye (PE-Cy7)-, or allophycocyanin (APC)-conjugated mAbs. Intracellular cytokine staining Cytofix/Cytoperm™ Kit and Foxp3/Transcription Factor Staining Buffer Set were purchased from BD Biosciences (San Jose, CA) and eBioscience (San Diego, CA), respectively, and analyses were performed according to the manufacturer’s instructions. The samples were collected and analyzed the data using BD Accuri C6 flow cytometer and software (BD Biosciences, San Jose, CA).
Kosaka A., Ohkuri T, & Okada H. (2014). Combination of an agonistic anti-CD40 monoclonal antibody and the COX-2 inhibitor celecoxib induces anti-glioma effects by promotion of type-1 immunity in myeloid cells and T-cells. Cancer immunology, immunotherapy : CII, 63(8), 847-857.
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5

Quantifying IL-2 Signaling Blockade

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Pan T cells were isolated from splenocytes using the Dynabeads FlowComp™ Mouse Pan T (CD90.2) kit. 200,000 mouse T cells and human PBMC, in complete RPMI were plated and rested for 2-3 hours at 37°C. Antibodies (mouse: αCD25PC61 or anti CD25NIB (both mIgG2a), αIL-2 neutralizing antibody (nAb) (JES6-1A12, BioXcell); human: RG6292, human IgG1 isotype control, Basiliximab (Absolute Antibody), Daclizumab (Absolute Antibody)) were added at 50 μg/ml (mouse) and 10 μg/ml (human), respectively, and were incubated with the cells for 30 mins at 37°C, following which cells were stimulated with IL-2 (50U/ml (mouse) and 10 U/ml (human) respectively, both Peprotech) for 10 mins at 37°C. IL-2 induced STAT5 phosphorylation was stopped when the cells were fixed and permeabilized with the eBioscience™ FoxP3 / Transcription Factor Staining Buffer Set and treated with the BD Phosflow Perm Buffer III. Blocking was calculated as follows: % blocking = 100 x [(% Stat5+ cells No Ab group - % Stat5+ cells 50ug/ml Ab group) / (% Stat5+ cells No Ab group)].
Solomon I., Amann M., Goubier A., Vargas F.A., Zervas D., Qing C., Henry J.Y., Ghorani E., Akarca A.U., Marafioti T., Śledzińska A., Sunderland M.W., Demane D.F., Clancy J.R., Georgiou A., Salimu J., Merchiers P., Brown M.A., Flury R., Eckmann J., Murgia C., Sam J., Jacobsen B., Marrer-Berger E., Boetsch C., Belli S., Leibrock L., Benz J., Koll H., Sutmuller R., Peggs K.S, & Quezada S.A. (2020). CD25-Treg-depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity. Nature cancer, 1(12), 1153-1166.
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6

Quantifying IL-2 Signaling Blockade

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Pan T cells were isolated from splenocytes using the Dynabeads FlowComp™ Mouse Pan T (CD90.2) kit. 200,000 mouse T cells and human PBMC, in complete RPMI were plated and rested for 2-3 hours at 37°C. Antibodies (mouse: αCD25PC61 or anti CD25NIB (both mIgG2a), αIL-2 neutralizing antibody (nAb) (JES6-1A12, BioXcell); human: RG6292, human IgG1 isotype control, Basiliximab (Absolute Antibody), Daclizumab (Absolute Antibody)) were added at 50 μg/ml (mouse) and 10 μg/ml (human), respectively, and were incubated with the cells for 30 mins at 37°C, following which cells were stimulated with IL-2 (50U/ml (mouse) and 10 U/ml (human) respectively, both Peprotech) for 10 mins at 37°C. IL-2 induced STAT5 phosphorylation was stopped when the cells were fixed and permeabilized with the eBioscience™ FoxP3 / Transcription Factor Staining Buffer Set and treated with the BD Phosflow Perm Buffer III. Blocking was calculated as follows: % blocking = 100 x [(% Stat5+ cells No Ab group - % Stat5+ cells 50ug/ml Ab group) / (% Stat5+ cells No Ab group)].
Solomon I., Amann M., Goubier A., Vargas F.A., Zervas D., Qing C., Henry J.Y., Ghorani E., Akarca A.U., Marafioti T., Śledzińska A., Sunderland M.W., Demane D.F., Clancy J.R., Georgiou A., Salimu J., Merchiers P., Brown M.A., Flury R., Eckmann J., Murgia C., Sam J., Jacobsen B., Marrer-Berger E., Boetsch C., Belli S., Leibrock L., Benz J., Koll H., Sutmuller R., Peggs K.S, & Quezada S.A. (2020). CD25-Treg-depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity. Nature cancer, 1(12), 1153-1166.
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7

Single-cell Immunophenotyping Workflow

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Single-cell suspensions were preincubated with anti-CD16/32 before staining with surface fluorescent conjugated antibodies and/or oligo-tagged antibodies (table S1) at predetermined concentrations in a 100 μl of staining buffer [phosphate-buffered saline (PBS) containing 5% fetal bovine serum (FBS) and 1% HEPES] per 107 cells. Stained cells were resuspended in 4′,6-diamidino-2-phenylindole (DAPI) in FACS buffer before purification or analysis. For intracellular staining, fixable Live/Dead dye was added before surface antibody incubation, which was performed according to the manufacturer’s instructions for the Foxp3/Transcription Factor Staining Buffer Set (BD Biosciences). Flow cytometry data were acquired on LSRII Analyzers (BD Biosciences) and then analyzed with FlowJo program. FACS was performed using FACSAria Cell Sorters (BD Biosciences).
Konieczny P., Xing Y., Sidhu I., Subudhi I., Mansfield K.P., Hsieh B., Biancur D.E., Larsen S.B., Cammer M., Li D., Landén N.X., Loomis C., Heguy A., Tikhonova A.N., Tsirigos A, & Naik S. (2022). Interleukin-17 governs hypoxic adaptation of injured epithelium. Science (New York, N.Y.), 377(6602), eabg9302.
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8

Multi-parameter flow cytometry analysis

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Bone marrow, blood, or splenocytes were processed and subjected to red blood cell lysis by ACK before counting via hemacytometer. Five million cells were resuspended in PBS and stained with Zombie Aqua viability dye (BioLegend, Cat# 423102) for 15 min at room temperature, covered from light. The cells were then washed with FACS buffer (PBS, 2% calf serum, 0.02% sodium azide) and resuspended in 25 µL 1:50 mouse FC block (TruStain FcX, BioLegend Cat# 101320), and left on ice for 5 min. 25 µL of a 2× cell surface staining antibody cocktail was added directly on top of the cells (final FC block 1:100, 1× Ab concentration) and stained on ice for 30 min. For intracellular staining, the cells were then washed with FACS buffer, permeabilized, and stained for intracellular targets according to the manufacturer’s protocol (eBioscience FOXP3 Transcription Factor Staining Buffer Set, Cat# 00-5523-00), then resuspended in FACS buffer before analyzing on either a BD Fortessa or Cytek Aurora flow cytometer. Data were analyzed using FlowJo software.
Romine K.A., MacPherson K., Cho H.J., Kosaka Y., Flynn P.A., Byrd K.H., Coy J.L., Newman M.T., Pandita R., Loo C.P., Scott J., Adey A.C, & Lind E.F. (2023). BET inhibitors rescue anti-PD1 resistance by enhancing TCF7 accessibility in leukemia-derived terminally exhausted CD8+ T cells. Leukemia, 37(3), 580-592.
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9

Flow Cytometry Analysis of T Cell Activation

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For flow cytometric analysis of T cells, the activation beads were removed using a magnet and the cells were washed and re-suspended in FACS buffer and stained with anti-CD25-BV786 (M-A251) (BD Bioscience, Herlev, Denmark) and Fixable Viability Dye (eFluor780, eBioscience, Waltham, MA, USA) for 30 min at 4 °C in the dark. Cells were permeabilized and fixed using the eBioscience FoxP3 Transcription Factor Staining Buffer Set (00-5523-00) according to manufacturer’s instructions and stained with anti-FoxP3-PE (236A/E7, BD Bioscience, Herlev, Denmark) for 30 min at room temperature in the dark. Samples were run on a five-laser BD LSRFortessa flow cytometer (BD Bioscience, Herlev, Denmark) and analyzed with FlowJo v10 software (Treestar, BD Bioscience, Herlev, Denmark).
Rode A.K., Buus T.B., Mraz V., Al-Jaberi F.A., Lopez D.V., Ford S.L., Hennen S., Eliasen I.P., Klewe I.V., Gharehdaghi L., Dragan A., Rosenkilde M.M., Woetmann A., Skov L., Ødum N., Bonefeld C.M., Kongsbak-Wismann M, & Geisler C. (2022). Induced Human Regulatory T Cells Express the Glucagon-like Peptide-1 Receptor. Cells, 11(16), 2587.
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10

Analyzing Th17, Th1, and Treg Cells in EAE Mice

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Mouse-specific anti-CD3 (Pacific Blue conjugated, clone 500A2, catalog no. 558214), anti-CD4 (PerCP conjugated, clone RM4-5, catalog no. 553052), anti-CD25 (Phycoerythrin/Cy7, clone PC61, catalog no. 561780), anti-FoxP3 (Alexa Fluor 488 conjugated, clone MF23, catalog no. 560403), anti-IL17A (Alexa Fluor 647 conjugated, clone TC11-18H10, catalog no. 560184), and IFNγ (PE conjugated, clone XMG1.2, catalog no. 554412) mAbs were all purchased from BD Biosciences.
For intracellular staining total splenocytes were isolated 37 days after EAE induction and cells were re-stimulated for 3 days with increasing amounts of MOG35-55. After that cells were stimulated for 4 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of GolgiPlug (1:1,000, BD Pharmingen). Cell were stained with surface markers (CD3, CD4, CD25), permeabilized using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set and stained for IL-17, IFNg, and FoxP3.
Samples were acquired on BD FACS Canto II flow cytometer and analyzed with FlowJo software.
The absolute count of Treg, Th1, and Th17 cells was measured as % singlets on 20,000 cells X % Treg or Th1 or Th17 on singlets/100.
Finardi A., Diceglie M., Carbone L., Arnò C., Mandelli A., De Santis G., Fedeli M., Dellabona P., Casorati G, & Furlan R. (2020). Mir106b-25 and Mir17-92 Are Crucially Involved in the Development of Experimental Neuroinflammation. Frontiers in Neurology, 11, 912.
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