Foxp3 fixation permeabilization buffer kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Foxp3 Fixation/Permeabilization Buffer Kit is a product designed for the intracellular staining and flow cytometric detection of Foxp3, a transcription factor that is critical for the development and function of regulatory T cells. The kit provides the necessary buffers to fix, permeabilize, and stain cells for the analysis of Foxp3 expression.

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Lab products found in correlation

Countbright beads, by Thermo Fisher Scientific (3 mentions) Live dead viability dye, by Thermo Fisher Scientific (3 mentions) Lsrfortessa flow cytometer, by BD (3 mentions) Lsrfortessa, by BD (3 mentions) Prism v8, by GraphPad (2 mentions) Foxp3 permeabilization buffer, by Thermo Fisher Scientific (2 mentions) Magnetic bead negative selection, by Miltenyi Biotec (1 mentions) Facsymphony, by BD (1 mentions) Ficoll paque, by GE Healthcare (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Lsrii instrument, by BD (1 mentions) Anti cd16 32 antibody 2.4g2 clone, by BioXCell (1 mentions) Fluorophore labeled antibodies, by Thermo Fisher Scientific (1 mentions) Celltrace cfse, by Thermo Fisher Scientific (1 mentions) Cd45ra bv605, by BioLegend (1 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions) Cd3 bv785 okt3, by BioLegend (1 mentions) Cd8 v500 rpa t8, by BD (1 mentions) Cd137 pecy7 4b4 1, by BioLegend (1 mentions) Cytofix cytoperm, by BD (1 mentions)

Spelling variants of this product

Permeabilization buffer (703 citations) Fixation/permeabilization buffer (268 citations) Fixation/permeabilization kit (189 citations) Foxp3 Fixation/Permeabilization kit (109 citations) Foxp3 Fixation/Permeabilization buffer (51 citations) Fixation/permeabilization buffer kit (22 citations) FoxP3 permeabilization buffer (21 citations) FoxP3 fixation buffer (19 citations) Foxp3 fixation kit (14 citations) FoxP3 buffer kit (6 citations)

11 protocols using foxp3 fixation permeabilization buffer kit

Isolation and Sorting of Tfh Cells

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Graft-draining axillary and brachial lymph nodes were processed into single-cell suspensions. Cells were surface stained with their appropriate markers followed by the fixable blue cell viability kit for UV excitation (LIVE/DEAD, Invitrogen) before fixation. Intracellular staining was performed with the use of the Foxp3 Fixation/Permeabilization Buffer Kit (eBioscience, Invitrogen). All antibodies were obtained from Biolegend and BD Biosciences. Samples were run on either LSR Fortessa or FACSymphony (BD Biosciences) and analyzed using FlowJo Software, version 10 (Flowjo, LLC). For sorting, cells were obtained from graft-draining axillary and brachial lymph nodes following skin transplantation. CD4+ T cells were enriched using magnetic bead negative selection (Miltenyi Biotec) and then sorted into CXCR5^– (CD19^–CD4⁺CD44^hiPD1^loCXCR5^–GITR^–) and CXCR5⁺ Tfh (CD19^–CD4⁺CD44^hiPD1^hiCXCR5⁺GITR^–) cell populations.

Crichton E.S., Zeng S., La Muraglia GM I.I, & Badell I.R. (2021). CXCL13 Is an Indicator of Germinal Center Activity and Alloantibody Formation Following Transplantation. Transplantation Direct, 7(12), e785.

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Comprehensive Lymph Node Cell Profiling

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Graft-draining axillary and brachial lymph nodes were processed into single cell suspensions. Cells were surface stained for CD3, CD4, CD19, CD44, CXCR5, PD-1, ICOS, Thy1.1, IgD, CD95 and GL-7, and pulsed with LIVE/DEAD viability dye (Molecular Probes) prior to fixation. CXCR5 staining was performed by one-step and three-step (30 (link)) techniques. Intracellular staining for Bcl6, CTLA-4 and Foxp3 was performed using the Foxp3 Fixation/Permeabilization Buffer Kit (eBioscience). All antibodies were purchased from BioLegend and BD Biosciences. All samples were run on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo Software (Tree Star). CountBright Beads (Invitrogen) were used to determine absolute cell counts.

Badell I., La Muraglia G., Liu D., Wagener M., Ding G, & Ford M. (2017). Selective CD28 blockade results in superior inhibition of donor-specific T follicular helper cell and antibody responses relative to CTLA-4-Ig. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 18(1), 89-101.

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Quantitative Lymphocyte Profiling

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Graft-draining axillary and brachial lymph nodes were processed into single-cell suspensions. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque (GE Healthcare) density gradient centrifugation of whole blood preparations. Cells were surface stained for indicated markers and pulsed with LIVE/DEAD viability dye (Molecular Probes) before fixation. Intracellular staining was performed with Foxp3 Fixation/Permeabilization Buffer Kit (eBioscience). All antibodies were from BioLegend and BD Biosciences. All samples were run on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed by using FlowJo Software (Flowjo, LLC). CountBright Beads (Invitrogen) were used to determine absolute cell counts.

La Muraglia GM I.I., Wagener M.E., Ford M.L, & Badell I.R. (2019). Circulating T Follicular Helper Cells Are a Biomarker of Humoral Alloreactivity and Predict Donor-Specific Antibody Formation After Transplantation. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 20(1), 75-87.

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Comprehensive Tumor Immune Profiling

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Single-cell preparations from the tumor were incubated with Live/Dead dye (Zombie NIR) to exclude the dead cells. After the wash, the cells were stained with a cocktail containing HPV16 E7 tetramer, CD3, CD4, CD8alpha, Foxp3, CD223 (Lag-3), CD279 (PD-1), and Hepatitis A virus cellular receptor 2 (also known as T-cell immunoglobulin and mucin-domain containing-3, Tim-3) antibodies for 30 min at 4°C. For detection of intracellular Foxp3, single cell suspensions were washed with FACS staining buffer, fixed, and permeabilized with a FoxP3 fixation/permeabilization buffer kit as per manufacturer’s instructions (eBioscience) before adding AF488-conjugated anti-mouse Foxp3 for 30 min at 4°C. Cells were acquired with a LSRII flow cytometer (BD biosciences) and analyzed with FlowJo software (Tree star, Ashland, OR, USA). In some experiments when the tumor volume measured less than 0.5 cm, single cell suspensions from individual mice of the same treatment group were combined to obtain a sufficient number of cells for flow analyses.

Ishida E., Lee J., Campbell J.S., Chakravarty P.D., Katori Y., Ogawa T., Johnson L., Mukhopadhyay A., Faquin W.C., Lin D.T., Wirth L.J., Pierce R.H, & Pai S.I. (2019). Intratumoral delivery of an HPV vaccine elicits a broad anti-tumor immune response that translates into a potent anti-tumor effect in a preclinical murine HPV model. Cancer immunology, immunotherapy : CII, 68(8), 1273-1286.

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Flow Cytometric Analysis of Immune Cell Populations

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For flow cytometric staining, fluorophore-labeled antibodies against the following markers were obtained from eBioscience (San Diego, CA, USA) unless otherwise indicated: CD4 (RM4-5), TCRβ (H57-597), CD8 (53-6.7), PD-1 (J43), CD5 (53-7.3), IL-7Rα/CD127 (A7R34), IFN-γ (XMG1.2), Bcl2 (10C4). Antibodies were used at manufacturer’s recommended concentrations. Flow cytometric staining always used an Fc block cocktail to block nonspecific staining. Fc block cocktail consisted of 3 mL each of normal mouse, rat, and hamster serum, with addition of 0.3 mg of anti-CD16/32 antibody (clone 2.4g2, BioXCell). For intracellular staining, cells were fixed and permeabilized using the eBioscience FoxP3 Fixation/permeabilization buffer kit according to supplied protocols. Standard flow cytometric analysis was performed using a BD LSR II instrument. Flow cytometric data analysis was performed using FlowJo (Treestar software, Portland, OR, USA).
Anti-IL-7Rα treatment in vivo was carried out by biweekly intraperitoneal injection of 0.5 mg of anti-IL-7Rα clone A7R34 (BioXcell for polyclonal PD-1^−/− thymocyte disease model experiments; or generated by us for mixed Marilyn experiments) or Rat IgG2a isotype control (2A3, BioXcell or generated by us) beginning on the day of cell transfer.

Ellestad K.K., Lin J., Boon L, & Anderson C.C. (2017). PD-1 Controls Tonic Signaling and Lymphopenia-Induced Proliferation of T Lymphocytes. Frontiers in Immunology, 8, 1289.

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Multiparametric flow cytometry of antigen-specific T cells

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Five million T-enriched cells were stained with a fixable live/dead stain (Aqua, Invitrogen) followed by phycoerythrin (PE)-coupled-2W:I-A^b tetramers for 1 hr in a room temperature (RT) water bath, washed, and then stained with PE-coupled-OVA:K^b pentamers (ProImmune, Oxford, UK) or PE-coupled-H60:K^b tetramers (NIH Tetramer Core Facility) for 20 min in a RT water bath. Cells were then stained with anti-CD4 (L3T4), anti-CD8 (Ly2), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD44 (IM7), anti-TCRβ (H57–597), and anti-B220 (RA3–6B2). Surface-stained cells were then fixed with the Foxp3 fixation permeabilization buffer kit (eBioscience, San Diego, California) for 15 min at RT and washed with 1 × permeabilization buffer. Some samples were intracellularly stained with anti-Ki-67 (SolA15), anti-IRF4 (3E4), or anti-Foxp3 (FJK-16 s) for 30 min at RT; washed with permeabilization buffer; and analyzed by flow cytometry. CFSE labeling was performed by labeling cells with CellTrace CFSE (Invitrogen) for 20 min at 37°C. All monoclonal antibodies (mAbs) were from BD Biosciences, eBioscience, or Invitrogen.

Miller M.L., McIntosh C.M., Williams J.B., Wang Y., Hollinger M.K., Isaad N.J., Moon J.J., Gajewski T.F., Chong A.S, & Alegre M.L. (2018). Distinct Graft-Specific TCR Avidity Profiles during Acute Rejection and Tolerance. Cell reports, 24(8), 2112-2126.

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Single-cell analysis of graft-draining lymph nodes

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Graft-draining lymph nodes were processed into single-cell suspensions. Cells were surface stained for indicated markers and pulsed with LIVE/DEAD viability dye (Molecular Probes) before fixation. Intracellular staining was performed with Foxp3 Fixation/Permeabilization Buffer Kit (eBioscience). All antibodies were from BioLegend and BD Biosciences. Samples were run on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed by using FlowJo Software (Flowjo, LLC). CountBright Beads (Invitrogen) were used to determine absolute cell counts.

La Muraglia GM I.I., Zeng S., Crichton E.S., Wagener M.E., Ford M.L, & Badell I.R. (2020). Superior inhibition of alloantibody responses with selective CD28 blockade is CTLA-4-dependent and T follicular helper cell-specific. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 21(1), 73-86.

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Multiparametric Phenotyping of Immune Cells

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Stimulated cells were stained with fluorochrome-conjugated monoclonal antibodies to detect surface and intracellular markers. Intracellular staining was performed with Foxp3 Fixation/Permeabilization buffer kit (eBioscience, San Diego, CA, USA), and Fixable Viability eFluor 780 Dye (eBioscience) was used to exclude dead cells. The following antibodies were used: CD4-Alexa Fluor 700 (OKT4, BioLegend), CD8-V500 (RPA-T8, BD Biosciences, San Jose, CA, USA), GrB-Fitc (GB11, BioLegend), IL-2-Pe (MQ1-17H12, BioLegend), IL-4-PeDaz594 (MP4-25D2, BioLegend), CD137-Pe/Cy7 (4B4-1, BioLegend), CD154-Alexa Fluor 647 (24-31, BioLegend), TNF-α-eFl450 (MAb11, eBioscience), IFN-γ-BV650 (4S.B3, BioLegend), CD3-BV785 (OKT3, BioLegend), CD19-BV605 (HIB19, BioLegend), CD45RA-BV605 (HI100, BioLegend), CCR7-PerCP/Cyanine 5.5 (G043H7, BioLegend).
For the absolute quantification of immune cell subsets, total CD19⁺, CD3⁺, CD4⁺, and CD8⁺ T cells numbers were determined in 50 μL of the peripheral blood by flow cytometry as described below by means of CytExpress software (Beckman Coulter, USA). Absolute numbers of HBs-specific T cells were determined based on relative frequencies of HBs-specific T cells and absolute numbers of total CD4⁺ T cells per μL of peripheral blood.

Awad G., Roch T., Stervbo U., Kaliszczyk S., Stittrich A., Hörstrup J., Cinkilic O., Appel H., Natrus L., Gayova L., Seibert F., Bauer F., Westhoff T., Nienen M, & Babel N. (2021). Robust hepatitis B vaccine-reactive T cell responses in failed humoral immunity. Molecular Therapy. Methods & Clinical Development, 21, 288-298.

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Multi-color flow cytometry of mouse and human T cells

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We conducted all flow staining for mouse and human T cells on ice and at room temperature, respectively. All mouse and human flow panel reagent information, stain conditions, and gating are included in (Supplemental Fig. 7, 8, 9Supplemental tables 1,2,3). We conducted LIVE/DEAD fixable aqua (AViD) staining in 1× PBS. For surface staining, we utilized FACSWash (1 × PBS supplemented with 2% FBS and 0.2% sodium azide) as the stain diluent. We fixed cells with the FOXP3 fixation/permeabilization buffer kit (Thermo Fisher) and conducted intranuclear stains using the FOXP3 permeabilization buffer (Thermo Fisher) as diluent. For ICS panels, we fixed cells with Cytofix/Cytoperm (BD Biosciences) and conducted intracellular stains using Perm/Wash buffer (BD Biosciences) as diluent. We resuspended cells in FACSWash and acquired events on a FACSSymphony, which we analyzed using FlowJo v10 (BD Biosciences). We conducted statistical testing using Prism v8 (GraphPad).

Taber A., Konecny A., Scott-Browne J, & Prlic M. (2023). TGF-β broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner. bioRxiv.

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Stimulation and Flow Cytometry Profiling

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Single suspension extracted cells, as described above, were plated into flow cytometry tubes (Sarstedt) at a concentration of 1 × 10⁶ per ml. Cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 μg/ml ionomycin, 2 μM monensin (all Sigma Aldrich) for 3–4 h as indicated. (Sigma Aldrich) FcR receptor-blocking antibodies were added before staining with antibodies. Surface staining antibodies were added with live/dead stain (Invitrogen). For intracellular staining, cells were fixed and permeabilised using the Foxp3 fixation/permeabilization buffer kit (Thermo Fisher) according to the manufacturer’s instructions. Samples were acquired using a BD LSRFortessa (BD Biosciences). Sample data was recorded in FCS 3.0 data format using BD FACSDiva 6.0 software (BD Biosciences). Analysis of the data was performed using FlowJo software (Treestar Inc., Ashland, OR, USA). Supplementary Table 3 provides the antibodies used and their dilutions.

Lo J.W., Cozzetto D., Alexander J.L., Danckert N.P., Madgwick M., Knox N., Sieh J.Y., Olbei M., Liu Z., Ibraheim H., Blanco J.M., Kudo H., Seoane R.C., Possamai L.A., Goldin R., Marchesi J., Korcsmaros T., Lord G.M, & Powell N. (2023). Immune checkpoint inhibitor-induced colitis is mediated by polyfunctional lymphocytes and is dependent on an IL23/IFNγ axis. Nature Communications, 14, 6719.

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