Fixation permeabilization kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Fixation/permeabilization kit is a laboratory tool designed to prepare samples for analysis. It enables the fixation and permeabilization of cells, allowing for the detection of intracellular markers. The kit contains the necessary reagents and instructions for this sample preparation process.

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Lab products found in correlation

Ionomycin, by Merck Group (14 mentions) Golgistop, by BD (12 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (12 mentions) Brefeldin a, by Thermo Fisher Scientific (12 mentions) Facscalibur, by BD (12 mentions) Leukocyte activation cocktail, by BD (10 mentions) Lsrfortessa, by BD (10 mentions) Phorbol myristate acetate (pma), by Merck Group (9 mentions) Brefeldin a, by BD (9 mentions) Anti cd4 fitc, by Thermo Fisher Scientific (8 mentions) Anti foxp3 apc, by Thermo Fisher Scientific (7 mentions) Golgiplug, by BD (7 mentions) Lsrii flow cytometer, by BD (6 mentions) Gallios flow cytometer, by Beckman Coulter (6 mentions) Facscalibur flow cytometer, by BD (6 mentions) Permeabilization buffer, by Thermo Fisher Scientific (6 mentions) Brefeldin a, by Merck Group (6 mentions) Lsrfortessa flow cytometer, by BD (6 mentions) Ack lysis buffer, by Thermo Fisher Scientific (5 mentions) Cytofix cytoperm kit, by BD (5 mentions)

Close spelling variants of this product

Fixation and permeabilization kit (29 citations) Intracellular fixation/permeabilization kit (10 citations) Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kit (9 citations) Fix&Perm Cell Fixation and Cell Permeabilization Kit (8 citations) IC Fixation/Permeabilization kit (8 citations) Fixation/Permeabilization Concentrate and Diluent kit (7 citations) Cell Fixation/Permeabilization Kit (5 citations) Intracellular fixation and permeabilization buffer kit (5 citations) Transcription Factor Fixation/Permeabilization kit (5 citations) Intracellular Fixation & Permeabilization Buffer kit (4 citations)

189 protocols using fixation permeabilization kit

Multiparametric Flow Cytometry Profiling

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Monoclonal antibodies (mAbs) to the following human proteins were used for staining: CD3 (UCHT1), CD4 (RPA-T4), IFN-γ (4S.B3), IL-17 (BL168) (Biolegend), phospho (p)-STAT1 (KIKSI0803), (all from eBioscience), STAT1 (246523; R&D Systems). Appropriate isotype controls were used in parallel. Peripheral blood mononuclear cells (PBMCs) were incubated with mAbs against surface markers for 30 min on ice. Intracellular staining with STAT1 mAb was performed using an eBioscience fixation/permeabilization kit according to the manufacturer’s instructions. For p-STAT1 staining, PBMCs were stimulated for 20 min with appropriate cytokines in complete medium, fixed with 2% paraformaldehyde for 20 min on ice, permeabilized with 90% methanol for 30 min on ice and stained using CD3, CD4 and p-STAT1 mAbs in PBS for 30 min. For cytokine detection, cell suspensions were incubated with Phorbol myristate acetate (PMA) (Sigma-Aldrich; 50 ng/mL), Ionomycin (Sigma-Aldrich; 500 ng/mL) and GolgiPlug™ (BD Biosciences; according to manufacturer’s instructions) for 4 h in complete medium before surface staining. Permeabilization and intracellular interferon (IFN)-γ and IL-17 staining was carried out using an eBioscience fixation/permeabilization kit as described above. Data were collected with an LSRFortessa™ cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Inc.).

Kiykim A., Charbonnier L.M., Akcay A., Karakoc-Aydiner E., Ozen A., Ozturk G., Chatila T.A, & Baris S. (2018). Hematopoietic Stem Cell Transplantation in Patients with Heterozygous STAT1 Gain-of-Function Mutation. Journal of clinical immunology, 39(1), 37-44.

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Phenotypic Analysis of Regulatory T Cells

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The phenotype of Treg cells was determined using 10⁶ spleen, mesenteric lymph nodes, or peritoneum cells. The following monoclonal antibodies were used for extracellular staining (all purchased from eBioscience, Affymetrix, Santa Clara, CA, unless stated otherwise): rat anti-mouse CD127 FITC (isotype rat IgG2a, κ) or hamster anti-mouse CD3ε FITC (isotype hamster IgG1, κ), rat anti-mouse CD4 Percp-Cy5.5 (isotype rat IgG2a, κ, BD Biosciences), and rat anti-mouse CD25 APC (isotype rat IgG1, κ, BD Biosciences). The cells were incubated with the antibodies for 30 min at 4°C. Then, the cells were left to permeabilize overnight with the Fixation/Permeabilization kit (Affymetrix). The cells were then washed, and intracellular staining was performed with rat anti-mouse Foxp3 PE (isotype rat IgG2a, κ), using the Fixation/Permeabilization kit (Affymetrix). The cells were incubated for 30 min at 4°C, washed, and fixed with (200 µL of 2%) paraformaldehyde in PBS.

Adalid-Peralta L., Lopez-Roblero A., Camacho-Vázquez C., Nájera-Ocampo M., Guevara-Salinas A., Ruiz-Monroy N., Melo-Salas M., Morales-Ruiz V., López-Recinos D., Ortiz-Hernández E., Demengeot J., Vazquez-Perez J.A., Arce-Sillas A., Gomez-Fuentes S., Parkhouse R.M., Fragoso G., Sciutto E, & Sevilla-Reyes E.E. (2021). Regulatory T Cells as an Escape Mechanism to the Immune Response in Taenia crassiceps Infection. Frontiers in Cellular and Infection Microbiology, 11, 630583.

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Characterizing Regulatory T Cells by Flow Cytometry

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Cells were washed in PBS, pelleted, and subsequently stained for flow cytometry. Treg cells were characterized accordingly with monoclonal antibodies against CD4, CD25, Foxp3 or CD4, Helios, Foxp3. To stain CD4, CD25, Foxp3 or CD4, Helios, Foxp3, anti-CD4-FITC (eBioscience, USA, #11-0042-81), anti-CD25-PE (eBioscience, USA, #35-0251-82), anti-Foxp3-APC (eBioscience, USA, #17-5773-82) or anti-CD4-Percp-cy5-5 (eBioscience, USA, #45-0042-82), anti-Helios-PE (eBioscience, USA, #12-9883-42), anti-Foxp3-FITC (eBioscience, USA, #11-4776-42), and a Fixation/Permeabilization kit (eBioscience, USA, 00-5123-43) were used according to the manufacturer’s instructions. At least 10⁵ cells were collected with a FACScan flow cytometer (Becton Dickinson) and analysed with Flow Jo software 7.6. Animal and cell flow experiments showed off the full gating once respectly and each performed the same gating for all analyses.

Wang C., Xie K., Li K., Lin S, & Xu F. (2021). Potential therapeutic effects of interleukin-35 on the differentiation of naïve T cells into Helios+Foxp3+ Tregs in clinical and experimental acute respiratory distress syndrome. Molecular Immunology, 132, 236-249.

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Intracellular TNFα Quantification

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Cells were seeded into non-treated tissue culture 24-well plates or round-bottom 96-well plates. The next day cells were stimulated with the indicated TLR ligands. To measure TNFα production, BrefeldinA (BD GolgiPlug, BD Biosciences) was added to cells 30 min after stimulation, and cells were collected after an additional 5.5 h. Dead cells were excluded using a fixable live/dead stain (Violet fluorescent reactive dye, Invitrogen). Cells were stained for intracellular TNFα with a Fixation & Permeabilization kit according to manufacturer’s instructions (eBioscience).
For flow cytometry on mouse cells, dead cells were excluded using a fixable live/dead stain (Aqua fluorescent reactive dye, Invitrogen) or DAPI and all stains were carried out in PBS containing 1% BSA (w/v) and 0.1% Azide (w/v) including anti-CD16/32 blocking antibody. Cells were stained for 20 min at 4°C with surface antibodies. Data were acquired on a LSRFortessa or X20 analyzer (BD Biosciences). See Extended Data Fig 10 for gating strategies.

Majer O., Liu B., Kreuk L.S., Krogan N, & Barton G.M. (2019). Unc93b1 recruits Syntenin-1 to dampen TLR7 signaling and prevent autoimmunity. Nature, 575(7782), 366-370.

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Measuring TLR-induced TNFα and DNA Uptake

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Cells were seeded into non-treated tissue culture 24-well plates or round-bottom 96-well plates. The next day cells were stimulated with the indicated TLR ligands. To measure TNFα production, BrefeldinA (BD GolgiPlug) was added to cells 30min after stimulation, and cells were collected after an additional 5.5h. Cells were stained for intracellular TNFα with a Fixation & Permeabilization kit according to manufacturer’s instructions (eBioscience).
For measuring DNA uptake, cells were fed Cy3-fluorescent CpG-B for the indicated amounts of time (control cells for no uptake were pre-chilled and stimulated on ice). Cells were washed 3 times with ice-cold PBS, fixed in 1% PFA/2%FCS/PBS, and analyzed on an BD LSR Fortessa flow cytometer.

Majer O., Liu B., Woo B.J., Kreuk L.S., Van Dis E, & Barton G.M. (2019). Release from Unc93b1 reinforces the compartmentalized activation of select TLRs. Nature, 575(7782), 371-374.

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Immune Cell Profiling with Intracellular Staining

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Cells were stained with the indicated antibodies (eBioscience and Biolegend). Lineage stain included antibodies to CD3, CD19, Gr-1, and FcεRIα. For intracellular cytokine staining, the Fixation & Permeabilization Kit (eBioscience) was used and cells were stained after a 5 hour restimulation period with 50 µg/mL PLP_139–151.

Russi A.E., Walker-Caulfield M.E., Ebel M.E, & Brown M.A. (2015). c-kit signaling differentially regulates ILC2 accumulation and susceptibility to CNS demyelination in male and female SJL mice. Journal of immunology (Baltimore, Md. : 1950), 194(12), 5609-5613.

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Comprehensive Immune Cell Analysis by Flow Cytometry

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Cells were stained with fluorescently tagged antibodies purchased from eBioscience, BD Biosciences, Tonbo Bioscience, or BioLegend (Supplementary Table 2) and analyzed using a BD LSR II flow cytometer. Flow cytometry data were analyzed using FlowJo software (TreeStar). For intracellular cytokine staining, cells were stimulated for 5 hr with CD3 and CD28 antibodies (5 μg/ml each) in the presence of brefeldin A or monensin, harvested and stained with eBioscience Fixation Permeabilization kit. For intracellular phosphorylated STAT5 staining, cells were stimulated with or without rmIL-2 for 20 min, fixed and permeabilized with 4% PFA followed by 90% methanol, and stained with anti-pY-STAT5 antibody (BD Biosciences). Cell sorting of Foxp3⁺ and Foxp3⁻ cells was performed based on YFP or GFP expression using a BD FACSAria II cell sorter.

Chinen T., Kannan A.K., Levine A.G., Fan X., Klein U., Zheng Y., Gasteiger G., Feng Y., Fontenot J.D, & Rudensky A.Y. (2016). An essential role for IL-2 receptor in regulatory T cell function. Nature immunology, 17(11), 1322-1333.

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Cytokine Analysis of CD4+ T-Cells

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Flow cytometric analysis was used to determine intracellular cytokines produced by the CD4⁺ T-cell clone 216-8E after 6 or 26 h of exposure to moDCs loaded with heat-inactivated TIGR4_ΔCPS (10⁷ culture-forming units/ml), PlyD1 (1 μg/ml), or no antigen as a control. In the last 4 h of stimulation, brefeldin A (BD Bioscience, San Jose, CA, USA) was added to capture intracellular cytokines. Cells were stained using fixable live/dead dye (ZombieNIR; BioLegend, San Diego, CA, USA) for 10 min at room temperature. After washing, surface proteins were stained using anti-human CD3 (SK7), CD4 (OKT4), and CD8 (SK1; all from BioLegend) antibodies for 20 min at 4°C. Cells were fixed and permeabilized using a fixation/permeabilization kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s protocol, followed by staining with anti-human IFN-γ (B27), TNF-α (Mab11), IL-17A (BL168) (all from BioLegend), and IL-4 (MP4-25D2; BD, Franklin Lakes, NJ, USA). Cells were measured using the fluorescence-activated cell sorter LSRFortessa X-20 (BD).

van de Garde M.D., van Westen E., Poelen M.C., Rots N.Y, & van Els C.A. (2019). Prediction and Validation of Immunogenic Domains of Pneumococcal Proteins Recognized by Human CD4+ T Cells. Infection and Immunity, 87(6), e00098-19.

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Multiparametric Flow Cytometry Analysis

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Lung and spleen cell staining was performed with CD16/CD32 Fc, CD4, CD25, CD31, CD326, CD45, F4/80 (Biolegend, San Diego, CA, USA), Ly6C, Ly6G, and CD11b (eBioscience, San Diego, CA, USA). Cells were fixed and permeabilized using a fixation/permeabilization kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Then, cells were stained for 30 min at 4°C with Foxp3 (Biolegend, San Diego, CA, USA) or Ki-67 (eBioscience, San Diego, CA, USA). The stained cells were washed twice and resuspended in 4% paraformaldehyde. Analysis of cell marker expression was performed using an Accuri C6 instrument (BD, Franklin Lakes, NJ, USA). Data were analyzed with FlowJo software.

Tan W., Zhang B., Liu X., Zhang C., Liu J, & Miao Q. (2021). Interleukin-33-Dependent Accumulation of Regulatory T Cells Mediates Pulmonary Epithelial Regeneration During Acute Respiratory Distress Syndrome. Frontiers in Immunology, 12, 653803.

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Flow Cytometry-based Immune Cell Profiling

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For flow cytometric analysis, single-cell suspensions were stained as described (25 (link)). Intracellular staining was conducted using a Fixation/Permeabilization kit (eBioscience, San Diego, CA, USA) following incubation of cells with Leukocyte Activation Cocktail (BD, San Diego, CA, USA) with Brefeldin A or Monensin (eBioscience). Fragment crystallizable (Fc) receptors were blocked using purified CD16/32 monoclonal antibody (mAb) (BD). Viable lymphocytes were assessed using Live/Dead Fixable Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA). Matching isotype mAbs were used to control for background staining.
Anti-mouse CD5 (53-7.3), B220 (RA3-6B2), CD3 (17A2) and CD39 (24DMS1) were purchased from eBioscience. Anti-mouse CD19 (6D5) was from Biolegend (San Diego, CA, USA). Anti-mouse CD4 (RM4-5), CD25 (PC61) and Foxp3 (MF23) were purchased from BD.
Human cells were stained with CD20 APC-Cy7 (L27) and CD5 PE-Cy7 (L17F12), (BD, San Diego, CA, USA). TLR9 PE (26C593.2) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). CD19 PE (HIB19) was purchased from eBioscience. A BD LSRII flow cytometer was used for assessment of cell suspensions (San Jose, CA, USA). Analysis was conducted using FlowJo software (Treestar, Ashland, OR, USA).

Saulep-Easton D., Vincent F.B., Quah P.S., Wei A., Ting S.B., Croce C.M., Tam C, & Mackay F. (2015). The BAFF receptor TACI controls IL-10 production by regulatory B cells and CLL B cells. Leukemia, 30(1), 163-172.

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