Transcription factor staining kit

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

The Transcription Factor Staining Kit is a laboratory tool designed for the detection and analysis of transcription factors within cells. It provides a standardized method for the preparation, fixation, and permeabilization of samples prior to immunostaining and flow cytometric analysis of transcription factor expression.

Automatically generated - may contain errors

Lab products found in correlation

Facscanto 2, by BD (3 mentions) Cytofix cytoperm kit, by BD (2 mentions) Lsrfortessa, by BD (2 mentions) Biotin anti p2ry12 antibody, by BioLegend (2 mentions) Streptavidin efluor450 conjugated antibody, by Thermo Fisher Scientific (2 mentions) Fortessa, by BD (2 mentions) Lsr 2, by BD (2 mentions) Anti pd 1 rmp1 30, by BioLegend (1 mentions) Eomes dan11mag, by Thermo Fisher Scientific (1 mentions) Facsaria instrument, by BD (1 mentions) Klrg1 2f1, by Thermo Fisher Scientific (1 mentions) Granzyme b gb12, by Thermo Fisher Scientific (1 mentions) T bet ebio4b10, by Thermo Fisher Scientific (1 mentions) Live dead fixable blue, by Thermo Fisher Scientific (1 mentions) Cytofix, by BD (1 mentions) Anti mouse tcr vβ screening panel, by BD (1 mentions) Facsdiva software, by BD (1 mentions) Anti cd16 32, by Thermo Fisher Scientific (1 mentions) Anti mouse lineage cocktail, by BioLegend (1 mentions) Accuri c6, by BD (1 mentions)

Spelling variants of this product

Transcription Kits (2 citations) Transcription factors staining kit (2 citations)

23 protocols using transcription factor staining kit

Comprehensive Immune Profile of T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Surface staining was performed for 30 min and with tetramer for 90 min at 4°C in PBS supplemented with 2% FCS and 0.01% azide (FACS buffer) using the following antibodies: anti-CD8α (53-6.7), CD127 (eBioSB/199), LAG-3 (C9B7W), and KLRG-1 (2F1; all eBioscience); anti–PD-1 (RMP1-30; BioLegend); gp276–286 tetramer (TCMetrix); and anti-CD4 (GK1.5), CD45.1 (A20), and CD45.2 (clone 104), obtained from or custom purified by Bio X Cell and coupled to Pacific blue, Alexa Fluor 647, or FITC using labeling reagents from Invitrogen. Cells were washed twice and fixed in PBS supplemented with 1% formaldehyde, 2% glucose, and 0.03% azide for 20 min. Then, cells were washed again and resuspended in FACS buffer. For intracellular cytokine staining, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD) and stained with anti–IFN-γ (XMG1.2), TNF (MP6-XT22; both from eBioscience), or granzyme B (GB12; Invitrogen). T-bet and Eomes staining was performed with a transcription factor staining kit (eBioscience) and stained with Eomes (Dan11mag) and T-bet (eBio4B10; both from eBioscience).
For flow cytometry sorting, living cells were stained in 10% FCS RPMI media and sorted on a FACSAria instrument (BD).

Utzschneider D.T., Alfei F., Roelli P., Barras D., Chennupati V., Darbre S., Delorenzi M., Pinschewer D.D, & Zehn D. (2016). High antigen levels induce an exhausted phenotype in a chronic infection without impairing T cell expansion and survival. The Journal of Experimental Medicine, 213(9), 1819-1834.

+ Open protocol

+ Expand

Multiparameter Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Freshly collected tissues were kept on ice, passed through a 40 µm filter then cells stained with a viability marker (dilution 1:1,000 in PBS; Live/Dead Fixable Blue, ThermoFisher Scientific). Cells were washed once in FACS buffer (0.5% BSA in PBS) then stained for surface antigens in FACS buffer for 30 min in the dark. Washed cells were fixed and permeabilized using Cytofix (BD Biosciences) for surface stains, or the Transcription Factor Staining kit (eBioscience) for intracellular stains. TCR Vβ repertoire was assessed using the anti‐mouse TCR Vβ Screening Panel (BD Biosciences). Data were acquired on a BD Fortessa and analyzed using FlowJo software (V.10, Treestar). Primary antibodies and their dilutions are listed under “Antibodies.”

Kuczynski E.A., Morlino G., Peter A., Coenen‐Stass A.M., Moss J.I., Wali N., Delpuech O., Reddy A., Solanki A., Sinclair C., Calado D.P, & Carnevalli L.S. (2022). A preclinical model of peripheral T‐cell lymphoma GATA3 reveals DNA damage response pathway vulnerability. EMBO Molecular Medicine, 14(6), e15816.

+ Open protocol

+ Expand

Characterization of Lung Immune Responses in WT and AhR-/- Mice

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

The lungs from P. brasilienis infected WT and AhR^−/− mice were collected after 96 h, 2 and 10 weeks of infection, digested enzymatically and lung leukocytes prepared as previously described^{39 (link)}. For cell-surface staining, lung cells were suspended at 1 × 10⁶ cells/mL in staining buffer. Fc receptors were blocked with unlabeled anti-CD16/32 (eBioscience) and then stained for 30 min on ice with fluorophore-conjugated antibodies. For myeloid cells the following antibodies were used: anti-CD11c, CD40, CD80, CD86 and MHC-II (IAb⁺). For lymphocytes: anti-CD4, CD25, CD8, CD44, and CD62L. For ILCs characterization, lung leukocytes were first treated with anti-mouse lineage cocktail (Biolegend) containing antibodies to CD3, Ly6G/Ly6C, CD11b, CD45R/B220, TER 119/erytroid cells, that react with T cells, B cells, monocytes, macrophages, NK cells and erythrocytes. Intracellular staining was conducted using the eBioscience Transcription Factor staining kit and specific antibodies for IL-17, IL-4, IFN-γ, IL, 22, IL-1β, IL-12, TNF-α, IL-6, TGF-β, IL-10, FoxP3, IDO-1 and AhR. Cells were run on FACSCantoII (BD Biosciences) and a minimum of 50,000 events was acquired using a FACSDiva software (BD Biosciences). Cells were analyzed using FlowJo software (Tree Star).

de Araújo E.F., Preite N.W., Veldhoen M., Loures F.V, & Calich V.L. (2020). Pulmonary paracoccidioidomycosis in AhR deficient hosts is severe and associated with defective Treg and Th22 responses. Scientific Reports, 10, 11312.

+ Open protocol

+ Expand

Multicolor Flow Cytometry Immunophenotyping

Check if the same lab product or an alternative is used in the 5 most similar protocols

To assess the expression of cell surface proteins, cells were incubated with fluorochrome-conjugated mAb at 4°C for 30 min. The cells were then washed with FACS buffer. For some experiments, the cells were then fixed with PBS containing 2% paraformaldeyhe. In procedures requiring intracellular staining, cells were permeabilized following surface staining using the transcription factor staining kit (eBioscience), stained for 1 h at 4°C with a second set of fluorochrome-conjugated mAb, and suspended in FACS buffer for acquisition. The fluorochrome-conjugated mAb used in surface and intracellular staining were as follows: B cell panel—FITC IgA, PerCP-eF710 IgM, AF594 IgG3, AF647 IgG2b, AF700 CD38, APC-eF780 “dump” (CD90.2, CD11c, F4-80, and GR1), BV510 IgE, BV605 IgG1, BV711 IgG2A, BV786 IgD, AF350 IgG (H+L), BUV395 B220; T cell surface panel –PE-Cy7 PD-1, AlexaFluor® (AF) 700 CD44, APC-eFluor® (eF) 780 “dump” (CD11b, CD11c, and B220), Brilliant Violet™ (BV) 421 CXCR5, BV650 CD8a, and Brilliant Ultraviolet™ (BUV) 395 CD4; and T cell ICS panel—AF488 Bcl6, BV605 Tbet, AF700 CD44, APC-eF780 “dump” (CD11b, CD11c, and B220), and BUV395 CD4.

Sjaastad F.V., Condotta S.A., Kotov J.A., Pape K.A., Dail C., Danahy D.B., Kucaba T.A., Tygrett L.T., Murphy K.A., Cabrera-Perez J., Waldschmidt T.J., Badovinac V.P, & Griffith T.S. (2018). Polymicrobial Sepsis Chronic Immunoparalysis Is Defined by Diminished Ag-Specific T Cell-Dependent B Cell Responses. Frontiers in Immunology, 9, 2532.

+ Open protocol

+ Expand

Multiparametric Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Single-cell suspensions were incubated with the Fc-receptor blocking reagent (Miltenyi Biotec) and then stained with immunofluorescence-conjugated mAbs following the manufacturer’s instructions (Table S5). For transcription factor staining, the cells were first surface stained before fixation and permeabilization using the transcription factor staining kit (eBioscience), followed by intranuclear staining. For intracellular cytokine staining, CD8⁺ T cells (1.0 × 10⁵ cells) were stimulated in vitro with 50 ng/mL Phorbol-12-myristat-13-acetate (PMA, Sigma-Aldrich) and 1 μg/mL Ionomycin (Sigma-Aldrich) for 5 h in the presence of 5 μg/mL GolgiPlug. Cell surface molecules were stained before fixation and permeabilization, using the fixation/permeabilization kit (BD Biosciences), followed by intracellular staining. Cell viability was determined using the Annexin V apoptosis detection kits (BioLegend), according to manufacturer’s instructions. The cell samples were analyzed on a flow cytometer (FACS Canto II or Accuri C6; BD Biosciences), and data were analyzed using FlowJo software v10.7 (Tree Star Inc.).

Mashima H., Zhang R., Kobayashi T., Hagiya Y., Tsukamoto H., Liu T., Iwama T., Yamamoto M., Lin C., Nakatsuka R., Mishima Y., Watanabe N., Yamada T., Senju S., Kaneko S., Idiris A., Nakatsura T., Ohdan H, & Uemura Y. (2020). Generation of GM-CSF-producing antigen-presenting cells that induce a cytotoxic T cell-mediated antitumor response. Oncoimmunology, 9(1), 1814620.

+ Open protocol

+ Expand

Comprehensive Single-Cell Analysis of Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Single immune cells were obtained from draining lymph nodes and tumor tissues of indicated mice. For cell surface analysis, a total of 1–5×10⁶ cells were stained with Abs in the dark at 4°C for 30 min. After washing with cold resuspension solution buffer (1×PBS supplemented with 2% FBS), cells were analyzed using CytoFLEX flow cytometer (Beckman Coulter). CytExpert software (V.2.4) was used for data analysis. To detect the expression of intracellular transcriptional factors, cells were fixed and permeabilized following 30 min surface staining according to the manual of Transcription Factor Staining kit (eBioscience), followed by anti-Foxp3 antibody staining and Flow Cytometry (FACS) analysis. For cytokine analysis, cell samples were stimulated in vitro with Phorbol 12-myristate 13-acetate/Ionomycin in the presence of Brefeldin A (BioLegend) and Monensin (BioLegend) for 4 hours. Cells were washed and stained with surface marker antibodies, fixed and permeabilized using Fixation/Permeabilization buffer (BioLegend), and stained with intracellular antibodies.

Zhang C., Lei L., Yang X., Ma K., Zheng H., Su Y., Jiao A., Wang X., Liu H., Zou Y., Shi L., Zhou X., Sun C., Hou Y., Xiao Z., Zhang L, & Zhang B. (2021). Single-cell sequencing reveals antitumor characteristics of intratumoral immune cells in old mice. Journal for Immunotherapy of Cancer, 9(10), e002809.

+ Open protocol

+ Expand

Multimodal Flow Cytometry Analysis of Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Single-cell suspensions were prepared from mouse spleen, bone marrow, inguinal lymph nodes, peritoneal cavity and blood. 1-4 x 10⁶ cells in PBS 2% FCS were transferred into appropriate wells of a 96-well U bottom plate. To prevent non-specific antibody binding, cells were incubated with F_c blocking antibody for 20 min at 4°C in the dark. Cells were then incubated with antibodies for 30 min, on ice and in the dark. To fix cells, they were incubated in 10% formalin (Sigma-Aldrich) for 15 min at 4°C, and washed and resuspended in PBS 2% FCS. To stain for intracellular nuclear proteins, cells were fixed and permeabilised using the manufacturer’s instructions and the eBioscience Transcription Factor Staining kit. Stained single-cell suspensions were acquired on the BD LSRFortessa™.
Where appropriate, following extracellular antibody staining, immune populations were sorted by fluorescence-activated cell sorting (FACS) on a FACS Aria III (BD Biosciences).

Masle-Farquhar E., Jeelall Y., White J., Bier J., Deenick E.K., Brink R., Horikawa K, & Goodnow C.C. (2023). CARD11 gain-of-function mutation drives cell-autonomous accumulation of PD-1+ ICOShigh activated T cells, T-follicular, T-regulatory and T-follicular regulatory cells. Frontiers in Immunology, 14, 1095257.

+ Open protocol

+ Expand

Multiparameter Flow Cytometry Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Single-cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. For live/dead discrimination, cells were stained with either LIVE/DEAD Blue Reactive Dye (Invitrogen) (20 minutes, on ice, before primary) or Alexa Fluor NHS Ester (ThermoFisher) (with primary stain), and 4μg/mL anti-CD16/32 (2.4G2; Bioxcell) to block Fc receptors. The following anti-mouse antibodies were used, with clone and source company listed: CD45 PerCP-Cyanine5.5 (30-F11, BioLegend), CD45 BV510 (30-F11, BioLegend), CX3CR1 BV605 (SA011F11, BioLegend), F4/80 BV711 (BM8, BioLegend), Ly6G PerCP-Cyanine5.5 (1A8, BioLegend), Ly6C PE/Cyanine7 (HK1.4, BioLegend), CD11b BUV395 (M1/70, BD Horizon), CD11c BV650 (N418, BioLegend), Ki67 PE/Dazzle (16A8, BioLegend). Intracellular Ki67 was stained using the eBioscience Transcription Factor Staining Kit, following the included protocol. The BD FACS ARIA II was used for cell sorting, and the Bio-Rad ZE5 Yeti was used for flow cytometry analysis. Data analysis was performed using FlowJo v10 (FlowJo LLC).

Kosugi A., Weissman A.M., Ogata M., Hamaoka T, & Fujiwara H. (1992). Instability of assembled T-cell receptor complex that is associated with rapid degradation of zeta chains in immature CD4+CD8+ thymocytes.. Proceedings of the National Academy of Sciences of the United States of America, 89(20), 9494-9498.

+ Open protocol

+ Expand

Multiparameter Immune Cell Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Equal numbers of cells were stained for each sample, washed with ice-cold PBS, and stained with Zombie Ultraviolet dye (BioLegend) for 10 min at room temperature. All samples were then blocked with 5 μg/ml αCD16/CD32 (2.4G2; BioLegend) in FACS buffer (PBS containing 2% FCS and 2 mM EDTA) before staining for specified surface markers at 4 °C for 25 min. For detection of intracellular molecules, following surface staining, cells were fixed with 1% paraformaldehyde in PBS for 10 min at room temperature, permeabilized with the Transcription Factor Staining Kit (eBioscience), and then stained with the anti-RELM-α FITC antibody (Peprotech, UK). Samples were acquired using a 5 laser Fortessa with BD FACSDiva software and analyzed with the FlowJo software (v9, Tree Star).

Bancroft A.J., Levy C.W., Jowitt T.A., Hayes K.S., Thompson S., Mckenzie E.A., Ball M.D., Dubaissi E., France A.P., Bellina B., Sharpe C., Mironov A., Brown S.L., Cook P.C., S. MacDonald A., Thornton D.J, & Grencis R.K. (2019). The major secreted protein of the whipworm parasite tethers to matrix and inhibits interleukin-13 function. Nature Communications, 10, 2344.

+ Open protocol

+ Expand

Immunophenotyping of Myeloid Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Single cell suspensions were stained with biotin anti-P2RY12 antibody (1:100, BioLegend) in Fc block for 20 min at 4°C, followed by streptavidin eFluor450 conjugated antibody (1:200, eBioscience) in FACS wash buffer. Intracellular IRF8 (IRF8-FITC/IRF8-APC, V3GYWCH, 1:100, eBioscience) was stained with eBioscience Transcription Factor staining kit. Cells were run on a FACSCanto II. Transduced cells were identified by GFP⁺. Data was analyzed using FlowJo.

Zhou Y., Song W.M., Andhey P.S., Swain A., Levy T., Miller K.R., Poliani P.L., Cominelli M., Grover S., Gilfillan S., Cella M., Ulland T.K., Zaitsev K., Miyashita A., Ikeuchi T., Sainouchi M., Kakita A., Bennett D.A., Schneider J.A., Nichols M.R., Beausoleil S.A., Ulrich J., Holtzman D.M., Artyomov M.N, & Colonna M. (2020). Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and - independent cellular responses in Alzheimer’s disease. Nature medicine, 26(1), 131-142.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!