Ebioscience foxp3 transcription factor staining kit

Manufactured by Thermo Fisher Scientific

The EBioscience™ Foxp3/transcription factor staining kit is a laboratory kit designed for the detection and analysis of transcription factors, including Foxp3, in cells. The kit provides the necessary reagents and protocols for the staining and flow cytometric analysis of intracellular transcription factors.

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Lab products found in correlation

Viability dye, by Thermo Fisher Scientific (1 mentions) Facs lsr fortessa flow cytometer, by BD (1 mentions) Trustain fcx, by BioLegend (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Golgiplug, by BD (1 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Anti cd69, by BioLegend (1 mentions) Anti cd98, by BioLegend (1 mentions) Anti cd25, by BioLegend (1 mentions) Anti cd71, by BioLegend (1 mentions) Anti cd8β, by BioLegend (1 mentions) Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (1 mentions) Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions) Lsrfortessa cell analyzer, by BD (1 mentions)

5 protocols using ebioscience foxp3 transcription factor staining kit

Phenotypic Analysis of Murine Tregs

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Splenocytes were stained with antibodies against surface markers (CD4, B220, CD41, and GARP) in the presence of a viability dye (eBioscience) and anti-CD16/32 to block FcγRs using a standard protocol. Tregs were stained with anti-Foxp3 using the eBioscience™ FOXP3/Transcription Factor Staining Kit (Invitrogen). Analyses were performed on a FACS LSR Fortessa flow cytometer (DIVA, BD Biosciences) and data were computed using the FlowJo software (Tree Star).

Gaignage M., Zhang X., Stockis J., Dedobbeleer O., Michiels C., Cochez P., Dumoutier L., Coulie P.G, & Lucas S. (2022). Blocking GARP-mediated activation of TGF-β1 did not alter innate or adaptive immune responses to bacterial infection or protein immunization in mice. Cancer Immunology, Immunotherapy, 71(8), 1851-1862.

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Tumor-Infiltrating Lymphocyte Profiling

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Tumors and lymph nodes were harvested. Cell suspensions were generated through mechanical tissue disruption and collagenase D digestion. Red blood cells were lysed, and samples were filtered through 60 μm nylon filters to obtain single cell suspensions. Cells were stained in PBS with TruStain fcX (BioLegend, Cat. No. 101320) to reduce non-specific antibody binding. Cell samples were then stained with anti-CD3-APC, CD4-FITC, and CD8-PECy5 conjugated antibodies (BioLegend, Cat. No. 100311, 100406, and 100709). After cell surface staining, cells were fixed and permeabilized (Invitrogen, eBioscience Foxp3/Transcription Factor Staining Kit, Cat. No. 00-5523-00) according to vendor’s protocol. Cells were then stained with anti-FoxP3-PE conjugated antibody. Samples were washed and analyzed with flow cytometry (Beckman Coulter CytoFLEX). Isotype control antibody-stained samples were used as negative staining controls. Flow cytometry data was analyzed using FlowJo Single Cell Analysis Software (Treestar, Inc., Ashland, Oregon) with gating strategies shown in Figure S10–12 (Supporting Information).

Zhou S., Zhen Z., Paschall A.V., Xue L., Yang X., Bebin-Blackwell A.G., Cao Z., Zhang W., Wang M., Teng Y., Zhou G., Li Z., Avci F.Y., Tang W, & Xie J. (2020). FAP-Targeted Photodynamic Therapy Mediated by Ferritin Nanoparticles Elicits an Immune Response against Cancer Cells and Cancer Associated Fibroblasts. Advanced functional materials, 31(7), 2007017.

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Cytokine Production in CD4 T Cells

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CD4 T cells were stimulated with PMA and Ionomycin (50 ng/ml and 1 μg/ml, respectively) (Sigma-Aldrich) for 5 hrs in the presence of GolgiPlug (BD Biosciences) at 1 μl/ml. Cells were then stained for CD4, TCRβ, CD44, and CD25 as described above. Afterwards, cells were fixed and permeabilized using the eBioscience^™ Foxp3/transcription factor staining kit (ThermoFisher). Cells were then stained with antibodies against IFNγ (clone XMG-1.2) and Tbet (clone ebio4B10).

Gaddis D.E., Padgett L.E., Wu R., Nguyen A., McSkimming C., Dinh H.Q., Araujo D.J., Taylor A.M., McNamara C.A, & Hedrick C.C. (2021). Atherosclerosis impairs naive CD4 T cell responses via disruption of glycolysis. Arteriosclerosis, thrombosis, and vascular biology, 41(9), 2387-2398.

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Cell Proliferation Analysis by Flow Cytometry

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To measure cell proliferation, cells were stained with a CellTrace Violet Cell Proliferation kit (Thermo Scientific) prior to culturing. Flow cytometry staining was undertaken in 96-well round-bottom plates with a minimum of 200,000 cells per well. To distinguish live cells, the cells were stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Thermo Scientific). For surface staining, the cells were incubated in Facs buffer (2.5% FBS and 0.05% sodium azide in PBS) with labeled antibodies, all Thermo Scientific unless otherwise indicated: anti-CD8β, anti-CD25, anti-CD69 (BioLegend), anti-CD71 (BioLegend) and anti-CD98. Intranuclear staining was done using the eBioScience Foxp3/Transcription Factor Staining kit (Thermo Scientific) and anti-Irf4, anti-T-bet, and anti-c-Myc (Cell Signaling) antibodies. Flow cytometry was performed on a MacsQuant Analyzer 10. Flow cytometry data were analyzed using FlowJo 10 software. Flow cytometry data were generated within the Flow Cytometry and Cell Sorting Facility in Ashworth, King's Buildings at the University of Edinburgh.

Toivakka M., Gordon K., Kumar S., Bermudez-Barrientos J.R., Abreu-Goodger C., Zamoyska R, & Buck A.H. (2024). miR-7 is recruited to the high molecular weight RNA-induced silencing complex in CD8+ T cells upon activation and suppresses IL-2 signaling. RNA, 30(1), 26-36.

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Isolation and Flow Cytometry of Colonic IELs

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Colonic IELs were obtained as previously described [9 (link)]. In brief, the colon was divided into 2-cm segments and incubated with Hank's Balanced Salt Solution +5 mM dithiothreitol for mucus removal. After two consecutive 10 min incubations with 5 mM EDTA, IELs were collected. LIVE–DEAD fixable aqua dead cell stain (ThermoFisher) was used to exclude dead cells from single-cell suspensions. Prior to flow cytometric analysis, suspensions were incubated in flow cytometry staining buffer with Anti-Mo CD16/CD32 (ThermoFisher, Clone93) for the blocking of non-specific binding of antibodies to Fc receptors. Staining for cell surface antigens was conducted using the antibodies listed in Table 3, and fixation was carried out using the eBioscience Foxp3/Transcription Factor Staining Kit (ThermoFisher). Flow cytometry was conducted using the LSRFortessa Cell Analyzer (BD Biosciences) running FACSDiva acquisition software. Data were analyzed using FlowJo v10.Table 3

List of Antibodies for Cell Sorting.

Table 3

Target	Fluorophore
CD3	PE-Cy7
TCRb	eFluor 450
CD8α	Alexa Fluor 700
CD8β	APC
CD4	APC-eFluor 780

Martchenko S.E., Martchenko A., Biancolin A.D., Waller A, & Brubaker P.L. (2021). L-cell Arntl is required for rhythmic glucagon-like peptide-1 secretion and maintenance of intestinal homeostasis. Molecular Metabolism, 54, 101340.

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