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Foxp3 fixation permeabilization kit

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The Foxp3 Fixation/Permeabilization kit is a laboratory reagent designed to facilitate the detection and analysis of intracellular proteins, specifically the transcription factor Foxp3. The kit provides the necessary reagents for the fixation and permeabilization of cells, allowing for the effective staining and flow cytometric analysis of Foxp3-expressing cells.

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Lab products found in correlation

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Spelling variants of this product

Fixation/permeabilization kit (189 citations) Foxp3 transcription factor fixation/permeabilization kit (44 citations) Foxp3 fixation kit (14 citations) Foxp3 Fixation/Permeabilization Buffer Kit (11 citations) Foxp3 Fixation/Permeabilization Concentrate and Diluent kit (10 citations) Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kit (9 citations) FoxP3 Fixation Permeabilization Staining Kit (4 citations) EBioscience Foxp3 fixation/permeabilization kit (3 citations) FoxP3 intracellular fixation and permeabilization kit (2 citations) EBioscience™ Foxp3/Transcription Factor Fixation/Permeabilization Kit (2 citations)

109 protocols using foxp3 fixation permeabilization kit

1

Intracellular Staining and Cryopreservation

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The cell pellet was resuspended in 100 μL of PBS and vortexed and 1 mL of 1× FOXP3 fixation buffer from FOXP3 Fixation/Permeabilization Kit (Thermo Fisher, 00-5523-00) was added, followed by thorough vortexing. After 30 min incubation at RT, 2 mL of 1× FOXP3 Permeabilization Buffer was added and the cells pelleted. The cell pellets were resuspended in 1 mL of 10% v/v DMSO (Sigma Aldrich, D2650) in CSM-I (Cell Staining Buffer—Intracellular), consisting of 5 mg/ml BSA and 0.2 mg/ml sodium azide in PBS, vortexed and stored at −20 °C until required.
Lee B.Y., Hogg E.K., Below C.R., Kononov A., Blanco-Gomez A., Heider F., Xu J., Hutton C., Zhang X., Scheidt T., Beattie K., Lamarca A., McNamara M., Valle J.W, & Jørgensen C. (2021). Heterocellular OSM-OSMR signalling reprograms fibroblasts to promote pancreatic cancer growth and metastasis. Nature Communications, 12, 7336.
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2

Flow Cytometry Analysis of Intracellular Proteins

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Surface staining for flow cytometry analysis was performed in PBS supplemented with 2% FBS at room temperature. Aqua Live/Dead (ThermoFisher, L34957) was used to exclude dead cells in all experiments. Intracellular protein staining was performed using the Foxp3 Fixation/Permeabilization kit (ThermoFisher, 00-5523-00) according to the manufacturer’s recommendation. For analysis of intracellular cytokine production following in vitro peptide stimulation, cells were fixed with 4% PFA in PBS and permeabilized with 1X Perm/Wash buffer (BD Bioscience, 554723). Antibodies and dilutions used for flow cytometry analysis are listed in Supplementary Fig. 6. Flow cytometry analysis was performed on a custom-order BD LSRFortessa instrument and analyzed using FlowJo v10.2 software (Treestar). Cell sorting was performed on a BD FACSAria Fusion instrument.
Waickman A.T., Victor K., Li T., Hatch K., Rutvisuttinunt W., Medin C., Gabriel B., Jarman R.G., Friberg H, & Currier J.R. (2019). Dissecting the heterogeneity of DENV vaccine-elicited cellular immunity using single-cell RNA sequencing and metabolic profiling. Nature Communications, 10, 3666.
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3

Multiparameter Flow Cytometry Analysis

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Cells were pre-incubated with 2.4G2 antibody (Bioceros) to block Fc receptors and stained with appropriate antibodies at 4°C in the dark for 30-45 minutes. Cell viability was assessed using Fixable Viability dyes (eFluor780 or eFluor506; Thermo Fischer) and cell suspensions were analyzed with a BD FACSymphony or purified using a BD Symphony S6, BD FACSAria II or III. Nuclei were sorted on basis of DAPI positivity and size. Analysis was performed with FlowJo software (BD). Intracellular staining for CD207 was performed by fixing and permeabilizing extracellularly stained cells according to the manufacturer’s instructions using the FoxP3 Fixation/Permeabilization Kit (Thermo Fischer).
Guilliams M., Bonnardel J., Haest B., Vanderborght B., Wagner C., Remmerie A., Bujko A., Martens L., Thoné T., Browaeys R., De Ponti F.F., Vanneste B., Zwicker C., Svedberg F.R., Vanhalewyn T., Gonçalves A., Lippens S., Devriendt B., Cox E., Ferrero G., Wittamer V., Willaert A., Kaptein S.J., Neyts J., Dallmeier K., Geldhof P., Casaert S., Deplancke B., ten Dijke P., Hoorens A., Vanlander A., Berrevoet F., Van Nieuwenhove Y., Saeys Y., Saelens W., Van Vlierberghe H., Devisscher L, & Scott C.L. (2022). Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches. Cell, 185(2), 379-396.e38.
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4

Multiparametric T cell analysis

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T cells were re-stimulated with phorbol myristate acetate (Sigma, 50 ng/mL) and ionomycin (Sigma, 500 ng/mL). 90 minutes later, cells were treated with Brefeldin A to block cytokine secretion. 3 h later, cells were stained for surface markers and simultaneously labelled with Live/Dead Blue Viability Dye (Thermo Fisher) for 20 minutes at 4°C. Cells were washed twice and fixed overnight using a FoxP3 Fixation/Permeabilization kit (Thermo Fisher). The following day, cells were washed and stained for intracellular cytokines at room temperature for 1 h. They were then washed 3 times and analysed using an LSR Fortessa machine (Beckman Dickinson). Analysis of mean fluorescence intensity was performed using FlowJo v10.0. All experiments were performed at least two independent times. Antibodies used (at 1:100 unless otherwise noted) were TNF-PE (BioLegend, MP6-XT22, 506306), PD-1-PECy7 (BioLegend, RMP1–30, 109110) IFN-γ-FITC (BioLegend, XMG1.2, 505806), CD4-BV711 (BioLegend, RM4–5, 100550), CD8α-BV786 (BioLegend, 53–6.7, 100750), Tox-PE (Miltenyi, REA473, 130–120-716, 1:50), Tcf7-Alexa647 (Cell Signaling, C63D9, 6709), PD-L1-APC (BioLegend, 10F.9G2, 124312, 1:5000), LAG-3-PerCP-Cy5.5 (BioLegend, C9B7W, 125211), TIM-3-PE-Dazzle594 (BioLegend, B8.2C12, 134013).
Vardhana S.A., Hwee M.A., Berisa M., Wells D.K., Yost K.E., King B., Smith M., Herrera P.S., Chang H.Y., Satpathy A.T., van den Brink M.R., Cross J.R, & Thompson C.B. (2020). Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nature immunology, 21(9), 1022-1033.
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5

Single Cell Staining and Analysis

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Single cell preparations were stained with antibodies against surface or intracellular markers as indicated in Supplementary Table 1. Specific conjugates used are indicated within Figures. Dead cells were excluded from analysis using the LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies). Intracellular staining was performed using the FoxP3 fixation/permeabilization kit (ThermoFisher). Samples were acquired using a BD Fortessa Cytometer, and analysed with FlowJo (TreeStar).
Penny H.A., Domingues R.G., Krauss M.Z., Melo-Gonzalez F., Lawson M.A., Dickson S., Parkinson J., Hurry M., Purse C., Jegham E., Godinho-Silva C., Rendas M., Veiga-Fernandes H., Bechtold D.A., Grencis R.K., Toellner K.M., Waisman A., Swann J.R., Gibbs J.E, & Hepworth M.R. (2022). Rhythmicity of Intestinal IgA Responses Confers Oscillatory Commensal Microbiota Mutualism. Science immunology, 7(75), eabk2541.
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6

Flow Cytometry Analysis of Murine Immune Cells

Cited 1 time
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Immune cells were isolated at time of euthanasia from P2 and P3 mouse blood by Ammonium-Chloride-Potassium (ACK) lysis of red blood cells at room temperature (63 (link)). Cells were washed with RPMI 1640 (Gibco), pelleted at 300g for 5 min at 4°C, and resuspended in magnetic-activated cell sorting (MACS) buffer [1× PBS (pH 7.2) + 2 mM EDTA + 0.5% BSA]. Cell suspensions were first stained with eBioscience Fixable Viability Dye eFluor 506 (Thermo Fisher Scientific/Invitrogen, catalog no. 65-0866-18) in PBS for 30 min at room temperature. Cells were stained with the following antimouse surface antibodies in MACS buffer for 30 min at room temperature: F4/80-BV785 (clone BM8; BioLegend, catalog no. 123141); CD45-BUV395 (clone 30-F11; BD Biosciences, catalog no. 564279); CD11b-FITC (clone M1/70; Thermo Fisher Scientific/Invitrogen, catalog no. 11-0112-41) or CD11b-PE (clone M1/70; BD Biosciences, catalog no. 557397); and Ly6G-APC (clone 1A8-Ly6g; Thermo Fisher Scientific/Invitrogen, catalog no. 17-9668-82). After surface antibody staining, the cells were fixed (30 min at room temperature) using the FoxP3 fixation/permeabilization kit (Thermo Fisher Scientific, catalog no. 00-5523-00). Stained cells were analyzed on a BD LSRFortessa (BD Biosciences) using the BD FacsDiva software (v9). Data were analyzed with BD FlowJo software version 10.9.
Joyce L.R., Kim S., Spencer B.L., Christensen P.M., Palmer K.L., Guan Z., Siegenthaler J.A, & Doran K.S. (2024). Streptococcus agalactiae glycolipids promote virulence by thwarting immune cell clearance. Science Advances, 10(22), eadn7848.
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7

Foxp3+ Regulatory T Cell Characterization

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T regs were cultured as described for 96 h, gently dissociated from the plate, and spun down at 5000 RPM for 10 min. Conditioned media was removed and saved for later use. Cells were fixed and permeabilized with the eBioscience FoxP3 fixation/permeabilization kit (Thermo Fisher Scientific #00-5521-00) according to manufacturer’s protocol. Cells were then taken up in flow buffer (1X PBS and 0.1% BSA), and stained with Foxp3 (Abcam Cat# ab218773), CD4 (AbCam Cat #ab269349), and CD25 (AbCam Cat# ab210330) for at least 30 min on ice protected from light. Cells were spun down; the supernatant was removed, and cells were washed in flow buffer once before being resuspended in 100 μL flow buffer. Flow cytometry was run on the Amnis FlowSight imaging flow cytometer and analysis was performed using IDEAS 6.2 software.
Farage P.K., McKee I.F, & Long S.P. (1998). Does a Low Nitrogen Supply Necessarily Lead to Acclimation of Photosynthesis to Elevated CO2?. Plant Physiology, 118(2), 573-580.
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8

Immune Cell Phenotyping Protocol

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Cell surface and intracellular staining were performed with monoclonal antibodies against CD3e (56-0032-82, Thermo Fisher Scientific, MA, USA) and CD4 (56-0032-82, Thermo Fisher Scientific). For intracellular staining, Foxp3 fixation/permeabilization kit (00-5523-00, Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Fluorescence intensities were examined as assessed by Attune NxT (Thermo Fisher Scientific) flow cytometer at the Icahn School of Medicine flow cytometry core, and data were analyzed using FCS express software (CA, USA).
Yang E.J., Rahim M.A., Griggs E., Iban-Arias R, & Pasinetti G.M. (2023). Transient anxiety-and depression-like behaviors are linked to the depletion of Foxp3-expressing cells via inflammasome in the brain. PNAS Nexus, 2(8), pgad251.
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9

Intracellular Cytokine Staining Assay

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SDLN or skin single cell suspensions were cultured with 1× Cell Stimulation Cocktail (Thermo Fisher Scientific). After 1 h, 1× Protein Transport Inhibitors) (500X) (Thermo Fisher Scientific) was added, followed by additional 4 h incubation at 37° C. The cells were then fixed, and permeabilized using FoxP3 fixation/permeabilization kit (Thermo Fisher Scientific) and stained intracellularly with anti-IFN-γ, IL-4, IL-17 or TNF-α for 60 min at 4° C.
Dai Z., Sezin T., Chang Y., Lee E.Y., Wang E.H, & Christiano A.M. (2022). Induction of T cell exhaustion by JAK1/3 inhibition in the treatment of alopecia areata. Frontiers in Immunology, 13, 955038.
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10

Foxp3+ Regulatory T Cell Characterization

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T regs were cultured as described for 96 h, gently dissociated from the plate, and spun down at 5000 RPM for 10 min. Conditioned media was removed and saved for later use. Cells were fixed and permeabilized with the eBioscience FoxP3 fixation/permeabilization kit (Thermo Fisher Scientific #00-5521-00) according to manufacturer’s protocol. Cells were then taken up in flow buffer (1X PBS and 0.1% BSA), and stained with Foxp3 (Abcam Cat# ab218773), CD4 (AbCam Cat #ab269349), and CD25 (AbCam Cat# ab210330) for at least 30 min on ice protected from light. Cells were spun down; the supernatant was removed, and cells were washed in flow buffer once before being resuspended in 100 μL flow buffer. Flow cytometry was run on the Amnis FlowSight imaging flow cytometer and analysis was performed using IDEAS 6.2 software.
Morin S.M., Gregory K.J., Medeiros B., Terefe T., Hoshyar R., Alhusseiny A., Chen S., Schwartz R.C., Jerry D.J., Vandenberg L.N, & Schneider S.S. (2022). Benzophenone-3 exposure alters composition of tumor infiltrating immune cells and increases lung seeding of 4T1 breast cancer cells. Advances in cancer biology - metastasis, 7, 100080.
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