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Foxp3 fixation kit

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The Foxp3 fixation kit is a laboratory product designed for the fixation and permeabilization of cells to facilitate the detection and analysis of intracellular Foxp3 protein expression. The kit provides the necessary reagents and protocols for the preparation of samples prior to flow cytometric analysis.

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Lab products found in correlation

Anti mouse cd16 cd32, by BioXCell (5 mentions) Software, by FlowJo (2 mentions) Lsrfortessa, by BD (2 mentions) Biotinylated human siglec 6 fc chimera, by R&D Systems (2 mentions) Bio protein l, by Thermo Fisher Scientific (2 mentions) Novocyte, by Agilent Technologies (2 mentions) Lsrii instrument, by BD (2 mentions) Attune nxt, by Thermo Fisher Scientific (2 mentions) Anti cd62l, by Thermo Fisher Scientific (1 mentions) Anti cd8a, by Thermo Fisher Scientific (1 mentions) Anti cd44, by Thermo Fisher Scientific (1 mentions) Anti il 17a, by Thermo Fisher Scientific (1 mentions) Anti t bet, by Thermo Fisher Scientific (1 mentions) Anti pakt s473, by Cell Signaling Technology (1 mentions) Anti p s6 s235 236, by Cell Signaling Technology (1 mentions) Brefeldin a, by BioLegend (1 mentions) Ionomycin, by Merck Group (1 mentions) Facscalibur, by BD (1 mentions) Facsaria 3, by BD (1 mentions) Lyse fix buffer, by BD (1 mentions)

14 protocols using foxp3 fixation kit

1

Intracellular Cytokine and Phospho-flow Analysis

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For intracellular cytokine staining, murine cells were restimulated after 72 h of culture with PMA (50 ng ml−1), Ionomycin (1 µg ml−1, both from Sigma-Aldrich) and brefeldin A (5 µg ml−1; Biolegend) for 4 h and fixed with 2% para-formaldehyde. Staining of transcription factors was performed without restimulation using the FOXP3/Fixation-Kit (eBioscience, 00–5521–00). Following antibodies were used for murine cell-analysis: anti-CD8a (eBiosciences, 53–6.7), anti-CD44 (eBiosciences, IM7), anti-CD62L (eBiosciences, MEL-14), anti-IFN-y (Biolegend, XMG1.2), anti-IL-17A (eBiosciences, eBio17B7), anti-RORyt (eBiosciences, B2D) or anti-T-BET (eBiosciences, eBio4B10). The cells were measured on FACSCalibur or FACSAria™ III (both BD) and analyzed with FlowJo Software (FlowJo LLC). For phospho-flow the cells were harvested after 48 h of culture, rested for 4 h and treated with 100U/ml rhIL-2 for 2 h, fixed and permeabilised using Lyse/Fix-Buffer (557870, BD) and Perm-BufferIII (558050, BD) then stained with anti-P-STAT5Y694 (eBiosciences, SRBCZX), anti-P-AKTS473 (Cell Signaling, D9E), anti-P-AKTY308 (Cell Signaling, D25E6), anti-P-FOXO1/3a (Cell Signaling, #9464) or anti-P-S6S235/236 (Cell Signaling, D57.2.2E).
Lückel C., Picard F., Raifer H., Campos Carrascosa L., Guralnik A., Zhang Y., Klein M., Bittner S., Steffen F., Moos S., Marini F., Gloury R., Kurschus F.C., Chao Y.Y., Bertrams W., Sexl V., Schmeck B., Bonetti L., Grusdat M., Lohoff M., Zielinski C.E., Zipp F., Kallies A., Brenner D., Berger M., Bopp T., Tackenberg B, & Huber M. (2019). IL-17+ CD8+ T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis. Nature Communications, 10, 5722.
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2

Identifying Antigen-Specific T Cells by Tetramer Enrichment

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Single cell suspensions were initially stained with GP66:I-Ab tetramer conjugated to Allophycocyanin for 1 hour at room temperature. Cells were then washed and incubated with anti-Allophycocyanin microbeads (Miltenyi Biotec) for 30 minutes. Tetramer positive cells were enriched as previously described (Moon et al.). The following surface stains were used: (purchased from BD Biosciences, Ebioscience, and Biolegend): anti-murine CD4 BV711 (RM4–5), anti-CD8 (BV510, BV650, BV711) (53–6.7), B220 PE-CF594 (RA3–6B2), anti-CD11b PE-CF594 (M1/70), anti-CD11c PE-CF594 (N418), anti-CD44-Alexa Fluor 700 (IM7), anti-CXCR5 PE-Cy7 (2G8), anti-PD1 (FITC, eFluor 450, BV605) (J43) anti-CD45.1 BV650 (A20), anti-CD45.2 FITC (104), anti-CD69 (FITC, Biotin, PE) (H1.2F3), , anti-CXCR3 BV421(CXCR3–173), anti-CD103-BV510 (M290), anti-Tbet-PE (4B10), FoxP3-eFluor450 (FJK-16s), and anti-RORγt-PE (AFKJS-9). Biotinylated antibodies were detected with Streptavidin BV605. Intracellular staining for Tbet and RORγt was performed using the Ebioscience FoxP3 fixation kit. All cells were run on the LSR II (BD) and analyzed using FlowJo software (Treestar).
Hondowicz B.D., Kim K.S., Ruterbusch M.J., Keitany G.J, & Pepper M. (2017). IL-2 is required for the generation of viral-specific CD4+ Th1 Trms cells and B cells are essential for maintenance in the lung. European journal of immunology, 48(1), 80-86.
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3

Intracellular Transcription Factor Staining

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After red blood cell lysis, total spleen cells were incubated with anti-mouse CD16/CD32 (BioXcell) for 5 minutes at RT, followed by surface staining for the indicated markers. For intracellular transcription factor staining, after surface markers were stained, cells were fixed and stained with antibodies against transcription factors by following Foxp3 fixation kit (eBioscience) instructions. Cell events were collected on an LSRII flow cytometer (Becton Dickonson).
Xie M.M., Koh B.H., Hollister K., Wu H., Sun J., Kaplan M.H, & Dent A.L. (2017). Bcl6 Promotes Follicular Helper T Cell Differentiation and PD-1 Expression in a Blimp1-independent Manner. European journal of immunology, 47(7), 1136-1141.
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4

Multiparametric T Cell Analysis

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Thymus and spleen were mechanically teased with glass slides to acquire single cell suspensions and cells were stained with antibodies to the following: CD45.1 (A20, eBioscience), CD45.2 (104, eBioscience), CD4 (RM4‐5, Biolegend), CD8 (53‐6.7, Biolegend), CD25 (PC61.5, Biolegend), CD44 (IM7, eBioscience), Qa2 (695H1‐9‐9, Biolegend), TCRβ (H57‐597, eBioscience), CD69 (H1.2F3, eBioscience), and H‐2Kb (AF6‐88.5, Biolegend). Reagents were conjugated to Pacific Blue, Brilliant Violet (BV) 421, BV510, BV711, PE, PE‐Cy7, PerCP–eFluor 710, allophycocyanin– eFluor 780 and Alexa Fluor 700. Streptavidin PE‐Cy7 (eBioscience) was used to detect biotinylated antibodies. A Foxp3 fixation kit (eBioscience) was used in conjunction with anti‐Foxp3 (FJK‐16s, eBioscience) or the BD Cytofix/Cytoperm Kit (BD Biosciences) to preserve the GFP signal. Data were acquired using a BD LSR Fortessa and FACSDiva 2.6 software and analysed using FloJo software (Tree Star).
Cowan J.E., Baik S., McCarthy N.I., Parnell S.M., White A.J., Jenkinson W.E, & Anderson G. (2018). Aire controls the recirculation of murine Foxp3+ regulatory T‐cells back to the thymus. European Journal of Immunology, 48(5), 844-854.
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5

Multiparametric Characterization of Immune Cells

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Single cell suspensions (PBS 2%FBS), were stained with fluorochrome-conjugated monoclonal antibodies against CD3 (OKT3 and SK7), CD4 (RPA-T4), CD8 (RPA-T8), CD19 (HIB19), CD5 (UCHT2), CD107a (H4A3), IFNγ (4S.B3), (BioLegend, San Diego, CA), CD327 (Siglec-6, clone REA852, Miltenyi Biotec, Bergisch Gladbach, Germany), biotinylated-human Siglec-6/Fc Chimera (R&D Systems, Minneapolis, MN) and bio-Protein L (Thermo Fisher Scientific, Waltham, MA) for 20 min at 4°C. For intracellular staining, cells were fixed and permeabilized with Foxp3 Fixation kit (eBioscience, San Diego, CA). Stained cells were acquired on an LSRII instrument (BD) or NovoCyte (ACEA Bioscience, San Diego, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR).
Kovalovsky D., Yoon J.H., Cyr M.G., Simon S., Voynova E., Rader C., Wiestner A., Alejo J., Pittaluga S, & Gress R.E. (2021). Siglec-6 is a Target for Chimeric Antigen Receptor T-cell Treatment of Chronic Lymphocytic Leukemia. Leukemia, 35(9), 2581-2591.
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6

Intracellular Transcription Factor Staining

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After red blood cell lysis, total spleen cells were incubated with anti-mouse CD16/CD32 (BioXcell) for 5 minutes at RT, followed by surface staining for the indicated markers. For intracellular transcription factor staining, after surface markers were stained, cells were fixed and stained with antibodies against transcription factors by following Foxp3 fixation kit (eBioscience) instructions. Cell events were collected on an LSRII flow cytometer (Becton Dickonson).
Wu H., Xie M.M., Liu H, & Dent A.L. (2016). Stat3 Is Important for Follicular Regulatory T Cell Differentiation. PLoS ONE, 11(5), e0155040.
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7

Immunophenotyping of Spleen Cells

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Cell suspension from spleen cells were firstly incubated with anti-mouse CD16/CD32 (Bio X Cell) for 5 min at rm temperature, followed with surface staining of indicated markers and viability staining. For intracellular staining, after surface staining, cell were fixed and stained with Abs against intracellular proteins by following Foxp3 fixation kit (eBioscience) protocol. Cell events were collected on LSRII flow cytometer (BD Biosciences) and analyzed by Flowjo® software.
Wu H., Chen Y., Liu H., Xu L.L., Teuscher P., Wang S., Lu S, & Dent A.L. (2016). Follicular regulatory T cells repress cytokine production by follicular helper T cells and optimize IgG responses in mice. European journal of immunology, 46(5), 1152-1161.
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8

Splenocyte Isolation and Multiparameter Flow Cytometry

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Mice were sacrificed, and spleens were harvested at the indicated time points after immunization. Splenocytes were isolated from red blood cells by ammonium-chloride-potassium (ACK) lysis buffer (Gibco) and resuspended in FACS buffer (5% FBS in 1× PBS). Cells were Fc blocked (clone 2.4G2; BD Biosciences) and stained with antibody master mix in FACS buffer for 30 min in the dark at 4°C. Secondary stains, where applicable, were also performed for 30 min in the dark at 4°C. After surface staining, cells were washed and fixed in Foxp3 fixation kit (eBioscience) for intranuclear staining or BD cytofix/cytoperm (BD Biosciences) for ICS or surface staining. Where applicable, intracellular markers were stained for 45 min to 1 h at 4°C. Samples were acquired on a BD LSRFortessa or BD FACSCelesta. All flow cytometry data were analyzed in FlowJo v10.
Lee J.H., Hu J.K., Georgeson E., Nakao C., Groschel B., Dileepan T., Jenkins M.K., Seumois G., Vijayanand P., Schief W.R, & Crotty S. (2020). Modulating the quantity of HIV Env-specific CD4 T cell help promotes rare B cell responses in germinal centers. The Journal of Experimental Medicine, 218(2), e20201254.
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9

Multiparametric Characterization of Immune Cells

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Single cell suspensions (PBS 2%FBS), were stained with fluorochrome-conjugated monoclonal antibodies against CD3 (OKT3 and SK7), CD4 (RPA-T4), CD8 (RPA-T8), CD19 (HIB19), CD5 (UCHT2), CD107a (H4A3), IFNγ (4S.B3), (BioLegend, San Diego, CA), CD327 (Siglec-6, clone REA852, Miltenyi Biotec, Bergisch Gladbach, Germany), biotinylated-human Siglec-6/Fc Chimera (R&D Systems, Minneapolis, MN) and bio-Protein L (Thermo Fisher Scientific, Waltham, MA) for 20 min at 4°C. For intracellular staining, cells were fixed and permeabilized with Foxp3 Fixation kit (eBioscience, San Diego, CA). Stained cells were acquired on an LSRII instrument (BD) or NovoCyte (ACEA Bioscience, San Diego, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR).
Kovalovsky D., Yoon J.H., Cyr M.G., Simon S., Voynova E., Rader C., Wiestner A., Alejo J., Pittaluga S, & Gress R.E. (2021). Siglec-6 is a Target for Chimeric Antigen Receptor T-cell Treatment of Chronic Lymphocytic Leukemia. Leukemia, 35(9), 2581-2591.
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10

Comprehensive Immune Cell Profiling

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After red blood cell lysis, total spleen cells were incubated with anti-mouse CD16/CD32 (BioXcell) for 5 minutes at RT, followed by surface staining for the indicated markers. For intracellular transcription factor staining, after surface markers were stained, cells were fixed and stained with antibodies against transcription factors by following Foxp3 fixation kit (eBioscience) instructions. Cell events were collected on an LSR2 flow cytometer (Becton Dickonson).
Wu H., Xu L.L., Teuscher P., Liu H., Kaplan M.H, & Dent A.L. (2015). An Inhibitory Role for the Transcription Factor Stat3 in Controlling IL-4 and Bcl6 Expression in Follicular Helper T cells. Journal of immunology (Baltimore, Md. : 1950), 195(5), 2080-2089.
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