Foxp3 fixation permeabilization buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The Foxp3 Fixation/Permeabilization buffer is a laboratory reagent used to prepare samples for flow cytometric analysis of intracellular proteins, such as the transcription factor Foxp3. The buffer is designed to fix and permeabilize cells, allowing for the staining and detection of intracellular targets.

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Foxp3 fixation/permeabilization buffer set (4 citations) Foxp3/Transcription Factor Fixation/Permeabilization Buffer (12 citations) Foxp3 staining fixation/permeabilization buffer (2 citations) Foxp3 Fixation/Permeabilization Buffer Kit (11 citations) Fixation/permeabilization buffer (268 citations) FoxP3 permeabilization buffer (21 citations) FoxP3 fixation buffer (19 citations) Permeabilization buffer (703 citations) Fixation buffer (92 citations)

51 protocols using foxp3 fixation permeabilization buffer

Intracellular Dlg1 and WASp Analysis in T Cells

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For intracellular Dlg1 and WASp analysis, T cells were fixed and permeabilized with the Foxp3 Fixation/Permeabilization buffer set (eBioscience 00-5523-00), washed and stained for 30 minutes at 4°C with primary antibodies (Dlg1 or WASp at 1:1000). Cells were washed, stained for 30 minutes at 4°C with Alexa Fluor647 AffiniPure F(ab)2 Donkey Anti-Mouse IgG (1:5000). Cells were then washed, fixed in 2% paraformaldehyde and analyzed with a BD FACS-Caliber.

Silva O., Crocetti J., Humphries L.A., Burkhardt J.K, & Miceli M.C. (2015). Discs Large Homolog 1 Splice Variants Regulate p38 –Dependent and –Independent Effector Functions in CD8+ T Cells. PLoS ONE, 10(7), e0133353.

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Multimodal T cell analysis protocols

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Immuno-staining for flow cytometric analysis was performed using the respective antibodies. For intracellular staining of cytokines, cells were treated with 1μg/ml phorbol 12-myristate 13-acetate (PMA), 1μg/ml ionomycin and 1μg/ml brefeldin A for two hours prior to staining. For Foxp3 staining, cells were fixed with Foxp3 fixation/permeabilization buffer (eBioscience, UK) for 30 minutes prior to staining, and if GFP was also detected, cells were fixed with 2% paraformaldehyde for five minutes prior to fixation/permeabilization. Data were acquired using a FACS Canto II (BD) and analyzed using FlowJo software (Tree Star).
Cytokine detection in culture supernatants from differentiated T cells was detected using the Flowcytomix^™ multiple analysis detection kit for Th1/Th2 cytokines (eBioscience, UK).
Western blot analysis was performed from total cell extracts prepared from naïve CD4⁺ T cells that were either un-activated or activated for 30 minutes with plate bound anti-CD3/anti-CD28 (1:2) with or without the addition of 2.5ng/ml TGF-β. Proteins were detected using the indicated antibodies and a horseradish peroxidase-conjugated secondary antibody and then visualized using the enhanced chemiluminescence detection system. Quantitation was performed by densitometric analysis of exposed films.

Singh Y., Garden O.A., Lang F, & Cobb B.S. (2015). MicroRNA-15b/16 enhances the induction of regulatory T cells by regulating the expression of Rictor and mTOR. Journal of immunology (Baltimore, Md. : 1950), 195(12), 5667-5677.

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PBMC Phenotypic Characterization by Flow Cytometry

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Peripheral blood mononuclear cells (PBMC) were isolated by density gradient
centrifugation in Ficoll Paque™ Plus (GE Healthcare Life Science, USA). PBMCs were
washed in phosphate-buffered saline and 0.5×10⁶ cells were incubated with
fluorescein isothiocyanate (FITC) or allophycocyanin (APC)-labeled anti-CD127,
APC-Cy3-labeled anti-CD3, peridinin chlorophyll (PerCP)-labeled anti-CD4, and
phycoerythrin (PE)-Cy7-labeled anti-CD25 antibodies (Becton Dickinson, USA),
according to the manufacturer's instructions. After 30 min of incubation at 4°C,
cells were washed with magnetic cell sorting (MACS) buffer, fixed and permeabilized
with FoxP3 fixation/permeabilization buffer (eBioscience, USA), and then processed
for FoxP3 staining using a kit containing APC-labeled anti-FoxP3 antibodies
(eBioscience) according to the manufacturer's instructions. PE-labeled anti-CTLA-4,
anti-PD1, anti-CD45RO, and anti-HLA-DR monoclonal antibodies and FITC-labeled
monoclonal antibodies to GITR, OX40, CD40L, CD28, and CD95 (Becton Dickinson) were
used for phenotypic evaluation of TREG and effector T cells following the same
labeling protocol described above. Cells were processed using a FACSCanto flow
cytometer (Becton Dickinson), and the acquired data were analyzed using the FlowJo
software (Tree Star Inc., USA).

Mesquita Júnior D., Cruvinel W.M., Araujo J.A., Salmazi K.C., Kallas E.G, & Andrade L.E. (2014). Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus. Brazilian Journal of Medical and Biological Research, 47(8), 662-669.

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Monensin-Mediated Protein Secretion Inhibition

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Protein secretion was inhibited in 1E06 cells by incubation with monensin (2 µM, 00-4505-51, Invitrogen) for 3 h at 37 °C. Intracellular Staining was performed following the Foxp3 Fixation/Permeabilization buffer set protocol (00-5523-00, eBioscience) and listed antibodies. mean fluorescent intensity (MFI) values were calculated using FlowJo.

Van Acker Z.P., Perdok A., Hellemans R., North K., Vorsters I., Cappel C., Dehairs J., Swinnen J.V., Sannerud R., Bretou M., Damme M, & Annaert W. (2023). Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism. Nature Communications, 14, 2847.

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Multiparametric Flow Cytometry Analysis

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Fc receptors were blocked with rat IgG (Jackson ImmunoResearch) and stained with the following antibodies: CD45 (30-F11), CCR9 (CW-1.2), CD45RB (C363-16A), CD44 (1M7), CD8 (53.6.7), CD19 (1D3), CD4 (RM4-5), CD25 (PC61.5), and GITR (DTA-1) from eBioscience, Lag3 (C9B7W) and CD62L (R1-2) from BD Bioscience and CCR4 (2G12), Tim3 (RMT3-23), H2D (34-2-12) from Biolegend). For intracellular staining, cells were fixed and permeabilized using the Foxp3 Fixation/Permeabilization buffer (eBioscience) and stained with CTLA-4 (UC10-4B9) and/or Foxp3 (FJK168) or fixed with Cytofix/Cytoperm (BD Biosciences) and stained with anti-BrdU according the manufacturer’s protocol (BrdU Flow Kit; BD Biosciences). Flow cytometric measurement of IL-2 secreting cells was conducted according to the manufacturer’s instructions (Mouse IL-2 Secretion Assay kit, Miltenyi Biotec).
Data were acquired on a FC-500 or Cytoflex flow cytometer (Beckman Coulter). Data were compensated and analyzed using WinList (Verity Software House) or FlowJo (Treestar) software. Division Index based on CFSE dilution was calculated using FCS Express 5 software (DeNovo) using the following equation:

\frac{\sum_{i = 1}^{P - 1} N_{i}}{\sum_{i = 1}^{P - 1} \frac{N_{i}}{2^{i}}}

P is the total number of peaks found and N is the number of cells in a generation. Fluorescence minus one controls were used for setting gates.

Ehrlich A.K., Pennington J.M., Tilton S., Wang X., Marshall N.B., Rohlman D., Funatake C., Punj S., O’Donnell E., Yu Z., Kolluri S.K, & Kerkvliet N.I. (2017). AhR activation increases IL-2 production by alloresponding CD4+ T cells initiating the differentiation of mucosal-homing Tim3+Lag3+ Tr1 cells. European journal of immunology, 47(11), 1989-2001.

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Quantitative Analysis of SMAD2/3 Cells

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The proportion of SMAD2/3-positive cells in control and S2/3-KO BxPC-3 cells was assessed by flow-cytometry. Control and S2/3-KO BxPC-3 cells were detached, washed and resuspended with Flow-Cytometry Staining Buffer (eBioscience #00-4222-26). To assess cell viability, cells were stained with LIVE/DEAD Far Red Dead Cell solution (Invitrogen #L10120). Cells were fixed and permeabilized for 30 min with 1x FoxP3 Fixation/Permeabilization Buffer (eBioscience #00-5523-00), washed, resuspended in 1x Permeabilization Buffer (eBioscience #00-8333-56), then blocked with 5%-FBS Permeabilization Buffer for 30 min. Cells were then incubated with anti-SMAD2/3 antibody for 45 min, washed twice with 1x Permeabilization Buffer, incubated with anti-rabbit Alexa Fluor 488-conjugated antibody (Invitrogen A32731) for 30 min and washed twice with 1x Permeabilization Buffer. Samples were resuspended in Flow-Cytometry Staining Buffer before analysis with BD Canto II flow cytometer. Results were interpreted with BD DIVA Software.

Bertrand-Chapel A., Caligaris C., Fenouil T., Savary C., Aires S., Martel S., Huchedé P., Chassot C., Chauvet V., Cardot-Ruffino V., Morel A.P., Subtil F., Mohkam K., Mabrut J.Y., Tonon L., Viari A., Cassier P., Hervieu V., Castets M., Mauviel A., Sentis S, & Bartholin L. (2022). SMAD2/3 mediate oncogenic effects of TGF-β in the absence of SMAD4. Communications Biology, 5, 1068.

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Identification and Quantification of Tregs

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Peripheral blood mononuclear cells were labeled with anti-CD4 (WSUMAB catalog #BOV2012) and anti-CD25 (WSUMAB catalog #BOV2076) primary antibodies and subsequently labeled with Tri-Color (Life Technologies SKU #M32006) and Alexa Fluor^® 488 (Life Technologies SKU #A-21151) as described previously. The eBiosciences FOXP3 fixation/permeabilization buffer was used to permeabilize cells according to manufacturer’s instructions, followed by 45 min incubation at 4°C in the dark with eBiosciences anti-mouse FOXP3-RPE antibody (clone FJK-16s). Rat IgG2a-RPE isotype was used as a control. Using a BD FACSCalibur Flow Cytometer, 50,000 events were collected and analyzed by using a subsequent gating strategy targeting lymphocytes, followed by CD4⁺ lymphocytes, then CD25^hi CD4⁺ lymphocytes, and finally FOXP3⁺CD25^hiCD4⁺ lymphocytes. These cells were then compared to the total number of lymphocytes present to determine the relative abundance of Tregs within a population.

Roussey J.A., Steibel J.P, & Coussens P.M. (2014). Regulatory T Cell Activity and Signs of T Cell Unresponsiveness in Bovine Paratuberculosis. Frontiers in Veterinary Science, 1, 20.

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Multiparametric Flow Cytometry Analysis

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Cells from lymph nodes and spinal cord were isolated as described before [26 ]. For detection of cell surface markers, cells were stained in FACS buffer (PBS containing 1% BSA and 0.1% NaN₃) with the following fluorochrome labeled monoclonal antibodies: anti-CD45 (30-F11), anti-CD4 (RM4-5), anti-CD25 (PC61) and anti-CD44 (IM7). For intracellular cytokine staining, cells were incubated for 16 hours with anti-CD3 (0.5 μg/ml). Next, cells were fixed and permeabilized by incubation with Foxp3 Fixation/Permeabilization Buffer (eBioscience) and stained in Permeabilization Buffer (eBioscience) with the following fluorochrome labeled monoclonal antibodies: anti-Foxp3 (FJK-16s), anti-IL-17 (eBio17B7) and anti-IFNγ (XMG1.2). All antibodies were purchased from BD Pharmigen or eBioscience. For cell number quantification, 10⁴ FACSuite FC Beads (BD) were added per sample prior to acquisition. Samples were acquired on FACS Verse (BD). FACS data were analyzed using FlowJo 7.6.5 software (TreeStar).

Koutrolos M., Berer K., Kawakami N., Wekerle H, & Krishnamoorthy G. (2014). Treg cells mediate recovery from EAE by controlling effector T cell proliferation and motility in the CNS. Acta Neuropathologica Communications, 2, 163.

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Multiparametric Flow Cytometry of T Cell Subsets

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Fluorochrome-conjugated antibodies specific for CD4, -CD8, -CD25, CD44 -IFN-γ, and -GATA3, CD62L, IL-2, IL-4, and Foxp3 were purchased from BD Biosciences (San Jose, CA) and eBiosciences (San Diego, CA). For nuclear staining, cells were fixed and permeabilized by FoxP3 fixation/permeabilization buffer (eBioscience) and stained with anti-Foxp3 or GATA3 antibody. Cells were analyzed using the FACSCantoII or LSRII flow cytometer (BD Biosciences) and data were analyzed using Flowjo software (Treestar, Ashland, OR).

Yu M., Owens D.M., Ghosh S, & Farber D.L. (2015). Conditional PDK1 ablation promotes epidermal and T cell-mediated dysfunctions leading to inflammatory skin disease. The Journal of investigative dermatology, 135(11), 2688-2696.

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Comprehensive Immune Cell Profiling

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Freshly isolated cells were stained on the same day as the isolation. For staining, cells were incubated with LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Fisher Scientific) and surface protein antibodies at 4 °C for 30 min. Stained cells were washed with FACS buffer and resuspended in Foxp3 Fixation/Permeabilization buffer (eBiosciences) according to manufacturer’s instructions. Fixed cells were stained for Bcl6 with PE anti-Bcl-6 (BD Biosciences) at room temperature for 30 mins. Cells were washed twice with 2 mL 1x permeabilization buffer before being resuspended in FACS buffer. Flow cytometric data were collected using a BD LSR Fortessa analyzer (BD Biosciences) with FACS Diva version 8.01 software. Data was subsequently analysed with FlowJo (version 10, Tree Star). Single leukocytes were identified based on forward and side scatter parameters, and expression of CD45. Dead and lineage positive cells were excluded using LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Fisher Scientific) and lineage markers (CD1a, CD14, CD34, CD94, BDCA2, FcεRIα, CD123, TCRγδ, CD19). T helped cells were gated at CD45⁺CD3⁺CD4⁺ before further gating as CXCR5⁺PD1⁺ TfH cells. ILCs were gated as Lin^-CD3^-CD127⁺ before further gating into CD117^-CRTH2^- ILC1s and CD117⁺CRTH2^- ILC3s, ILC3s were then gated as NKp44⁺NRP1⁺ or NKp44^-NRP1^-.

Tachó-Piñot R., Stamper C.T., King J.I., Matei-Rascu V., Richardson E., Li Z., Roberts L.B., Bassett J.W., Melo-Gonzalez F., Fiancette R., Lin I.H., Dent A., Harada Y., Finlay C., Mjösberg J., Withers D.R, & Hepworth M.R. (2023). Bcl6 is a subset defining transcription factor of Lymphoid Tissue inducer-like ILC3. Cell reports, 42(11), 113425.

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