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Transcription factor staining buffer kit

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The Transcription Factor Staining Buffer Kit is a set of reagents designed for the detection and analysis of transcription factors in cells. The kit provides the necessary buffers and solutions to prepare samples and stain transcription factors for flow cytometry or other applications.

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Lab products found in correlation

Cx3cr1, by BD (2 mentions) Klrg1 2f1, by BD (2 mentions) Thy1.1 ox 7 or his51, by BD (2 mentions) Fixable viability stain, by BD (2 mentions) Lsrfortessa, by BD (2 mentions) Golgiplug, by BD (2 mentions) Cytofix, by BD (2 mentions) Diva software, by BD (2 mentions) Live dead fixable cell stain, by Thermo Fisher Scientific (1 mentions) Facsymphony, by BD (1 mentions) Lsr fortessa 2, by BD (1 mentions) Tryple, by Thermo Fisher Scientific (1 mentions) 40 μm cell strainer, by BD (1 mentions) Protein transport inhibitor, by Thermo Fisher Scientific (1 mentions) Facscan ow cytometer, by BD (1 mentions) Tim3 pe cy7, by BD (1 mentions) Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions) Cd8 percp, by BD (1 mentions) Cd3 apc, by BD (1 mentions) Cd45 apc cy7, by BD (1 mentions)

Close spelling variants of this product

EBioscience™ FoxP3/Transcription Factor Staining Buffer Kit Transcription Factor Staining Buffer Set kit Foxp3/Transcription Factor Staining Buffer Kit Foxp3/Transcription Factor Staining Buffer Set Kit Transcription factor staining buffer Transcription buffer Staining buffer Transcription Kits

8 protocols using transcription factor staining buffer kit

1

Comprehensive Immune Phenotyping of CD8+ T Cells

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Single-cell suspensions were plated and stained for 20 min at 4 °C with a combination of fluorescently labeled antibodies specific for surface markers including CD8α (53–6.7), CD25 (PC61.5), CD11a (M17/4), CD43glyco (1B11), CD44 (IM7) CD49a (Ha31/8), CD62L (MEL-14), CD69 (H1.2F3), CD103 (2E7), CD122 (5H4), CXCR3 (CXCR3–173), CXCR6 (SA051D1), CX3CR1 (SA011F11), KLRG1 (2F1), Thy1.1 (OX-7 or HIS51) and fixable viability stain (BD Horizon). Antibodies were purchased from BD Bioscience, BioLegend, eBioscience/Thermo Fischer Scientific, or Tonbo biosciences. Cells were fixed with BD Cytofix (BD Biosciences).
To asses cytokine production, single cells suspensions were plated in the absence (no stim) or in the presence 1 μM OVA(257–264) peptide in the presence of GolgiPlug (BD Bioscience) for 5 h at 37 °C or detected directly ex vivo in the absence of GolgiPlug. Cells were stained for surface molecules including fixable viability stain (BD Horizon), fixed and permeabilized using the transcription factor staining buffer kit (Thermo Fisher Scientific) and stained for cytokines including IFN-γ (XMG1.2), TNF (MP6-XT22), and IL-2 (JES6–5H4).
All samples were acquired using LSRFortessa (BD Bioscience) and analyzed using FlowJo software, version 9.9.4 (FlowJo LLC).
Urban S.L., Jensen I.J., Shan Q., Pewe L.L., Xue H.H., Badovinac V.P, & Harty J.T. (2020). Peripherally induced brain tissue-resident memory CD8+ T cells mediate protection against CNS infection. Nature immunology, 21(8), 938-949.
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2

Comprehensive Immune Phenotyping of CD8+ T Cells

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Single-cell suspensions were plated and stained for 20 min at 4 °C with a combination of fluorescently labeled antibodies specific for surface markers including CD8α (53–6.7), CD25 (PC61.5), CD11a (M17/4), CD43glyco (1B11), CD44 (IM7) CD49a (Ha31/8), CD62L (MEL-14), CD69 (H1.2F3), CD103 (2E7), CD122 (5H4), CXCR3 (CXCR3–173), CXCR6 (SA051D1), CX3CR1 (SA011F11), KLRG1 (2F1), Thy1.1 (OX-7 or HIS51) and fixable viability stain (BD Horizon). Antibodies were purchased from BD Bioscience, BioLegend, eBioscience/Thermo Fischer Scientific, or Tonbo biosciences. Cells were fixed with BD Cytofix (BD Biosciences).
To asses cytokine production, single cells suspensions were plated in the absence (no stim) or in the presence 1 μM OVA(257–264) peptide in the presence of GolgiPlug (BD Bioscience) for 5 h at 37 °C or detected directly ex vivo in the absence of GolgiPlug. Cells were stained for surface molecules including fixable viability stain (BD Horizon), fixed and permeabilized using the transcription factor staining buffer kit (Thermo Fisher Scientific) and stained for cytokines including IFN-γ (XMG1.2), TNF (MP6-XT22), and IL-2 (JES6–5H4).
All samples were acquired using LSRFortessa (BD Bioscience) and analyzed using FlowJo software, version 9.9.4 (FlowJo LLC).
Urban S.L., Jensen I.J., Shan Q., Pewe L.L., Xue H.H., Badovinac V.P, & Harty J.T. (2020). Peripherally induced brain tissue-resident memory CD8+ T cells mediate protection against CNS infection. Nature immunology, 21(8), 938-949.
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3

Intracellular Protein Staining Protocol

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The transcription factor staining buffer kit (eBioscience) is used for the fixation, permeabilization, and staining of intracellular proteins using the following procedure:
Kastenschmidt J.M., Avetyan I, & Villalta S.A. (2018). Characterization of the Inflammatory Response in Dystrophic Muscle Using Flow Cytometry. Methods in molecular biology (Clifton, N.J.), 1687, 43-56.
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4

Comprehensive Evaluation of Rhesus Macaque Lung Granulomas

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Rhesus macaque BAL samples were passed through a 100-μm cell strainer, pelleted, and counted for analysis. Granulomas were individually resected from the lungs, and samples used for flow cytometry analysis were pushed through a 100-μm cell strainer. Aliquots from all samples were serially diluted and plated on 7H11 agar plates for CFU quantification. Alternatively, resected granulomas were fixed in 4% paraformaldehyde for later paraffin embedding. Samples were stained with surface antibody cocktails for 20 min at 4°C followed by fixable live/dead cell stain (Molecular Probes; Invitrogen). Cells were then fixed and permeabilized (eBioscience Transcription Factor Staining Buffer Kit) for at least 1 h followed by intracellular antigen staining for 30 min at 4°C. Cells were washed and samples acquired on a FACSymphony (BD Biosciences) at NIH. FACS data were analyzed using FlowJo 10 (FlowJo, LLC).
Bohrer A.C., Castro E., Hu Z., Queiroz A.T., Tocheny C.E., Assmann M., Sakai S., Nelson C., Baker P.J., Ma H., Wang L., Zilu W., du Bruyn E., Riou C., Kauffman K.D., Moore I.N., Del Nonno F., Petrone L., Goletti D., Martineau A.R., Lowe D.M., Cronan M.R., Wilkinson R.J., Barry CE I.I.I., Via L.E., Barber D.L., Klion A.D., Andrade B.B., Song Y., Wong K.W, & Mayer-Barber K.D. (2021). Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice. The Journal of Experimental Medicine, 218(10), e20210469.
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5

Pancreas Dissociation and Flow Cytometry

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The pancreas was dissected before dissociation in TrypLE (Invitrogen) at 37 °C for 30 minutes with agitation. The cells were quenched in FACS buffer (2% FBS, 10 mM EDTA in PBS) and filtered through a 40 μm cell strainer (BD Biosciences). The cells were stained using transcription factor staining buffer kit (Invitrogen). The cells were incubated in fixation and permeabilization buffer on ice for 15 minutes, washed with wash buffer, and stained with primary antibodies for 1 h on ice in wash buffer. After another wash, the cells were stained with secondary antibodies for 1 h on ice in wash buffer. The cells were spun down, resuspended in FACS buffer (3% FBS in PBS), and analyzed on LSRFortessa II (BD Biosciences). Data were analyzed with FlowJo software (Tree Star Inc.).
Scavuzzo M.A., Hill M.C., Chmielowiec J., Yang D., Teaw J., Sheng K., Kong Y., Bettini M., Zong C., Martin J.F, & Borowiak M. (2018). Endocrine lineage biases arise in temporally distinct endocrine progenitors during pancreatic morphogenesis. Nature Communications, 9, 3356.
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6

Quantifying Immune Inhibitory Molecules in Tumor CD8+ T Cells

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To quantify levels of immune inhibitory molecules of CD8 + T cells in tumor specimens, single cell suspensions were surface or intracellular stained with the following uorescent antibodies:CD45-APC-cy7, CD3-APC, CD8-PerCP, TIM3-PE-Cy7, BATF-PE, NR4A1-FITC, IFN--BV510(BD Biosciences,Germany).
For BATF,NR4A1 staining, cells were xed and permeabilized with the Transcription Factor staining buffer kit(Invitrogen, United States)according to the manufacturer's instructions. For IFN-staining, single-cell suspensions were rst stimulated with cell stimulation cocktail(500× (Invitrogen, United States and inhibited with protein transport inhibitor(500× (Invitrogen, United States), and then xed and permeabilized with Cyto x/Cytoperm Fixation/Permeabilization Solution Kit(BD Biosciences,Germany).Stained samples were collected and analyzed using a FACScan ow cytometer (BD Biosciences,Germany). Finally, all the data were analyzed by the FlowJo software (v10,United States).
Mi L., Gong D., Wang L., Liang X., Jiao M., Huang W., & Zhou J. (2021). PAX5 Haploinsufficiency Induces Immune Inhibitory-Related Molecules Expressions of CD8 + T Cells in the Tumor Microenvironment.
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7

Comprehensive PBMC Immunophenotyping Protocol

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Fresh or cultured PBMCs were stained with antibodies against surface markers, including CD3-APC, CD4-PE/Cy7, CD8-APC/Cy7, CD28-PE, CD127-PerCP/Cy5.5. Then intracellular staining was performed using a transcription factor staining buffer kit (Thermo Fisher Scientific-eBioscience; San Diego, CA, USA), according to the manufacturer’s instructions. After fixation and permeabilization, cells were incubated with antibodies against intracellular markers including GZMB (granzyme B)-FITC and Ki67-PerCP/Cy5.5 for 30 minutes. The mean fluorescence intensity (MFI) of GZMB was calculated. All fluorescent antibodies were purchased from BioLegend (San Diego, CA, USA). Samples were analyzed on FACSCanto using Diva software (BD Biosciences; San Jose, CA, USA).
Xia C.S., Long Y., Liu Y., Alifu A., Zeng X, & Liu C. (2022). IL-7 Promotes the Expansion of Circulating CD28- Cytotoxic T Lymphocytes in Patients With IgG4-Related Disease via the JAK Signaling. Frontiers in Immunology, 13, 922307.
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8

Flow Cytometry Analysis of PBMC Subsets

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Peripheral blood mononuclear cells (PBMCs) were separated by gradient centrifugation with a human lymphocyte separation medium (Dakewei Biotech Co., Ltd.; Shenzhen, China) and then washed twice with PBS. Enriched PBMCs were immediately stained for 30 minutes with the following antibodies: CD3-APC, CD4-PerCP/Cy5.5, CXCR5-APC/Cy7, CXCR3-PE, and CCR6-PE/Cy7. Intracellular staining for Foxp3 was performed using a transcription factor staining buffer kit (Thermo Fisher Scientific-eBioscience; San Diego, CA, USA), according to the manufacturer's instructions. After fixation and permeabilization, cells were incubated with anti-Foxp3 allophycocyanin for 30 minutes. All fluorescent antibodies were purchased from BioLegend (San Diego, CA, USA). Samples were analyzed on FACSCanto using Diva software (BD Biosciences; San Jose, CA, USA). Based on the number of lymphocytes in the complete blood count and the proportion of each subgroup of lymphocytes determined by flow cytometry, the absolute count (per μL) of each subgroup was calculated.
Xia C., Liu C., Liu Y., Long Y., Xu L, & Liu C. (2020). Increased Circulating Th1 and Tfh1 Cell Numbers Are Associated with Disease Activity in Glucocorticoid-Treated Patients with IgG4-Related Disease. Journal of Immunology Research, 2020, 3757015.
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