Fixation permeabilisation buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Fixation/permeabilisation buffer is a laboratory reagent used to fix and permeabilize cells prior to various analytical procedures, such as flow cytometry and immunofluorescence microscopy. The buffer helps to preserve the cellular structure and allow for the efficient penetration of antibodies or other probes into the cells for detection of intracellular targets.

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EBioscience™ Intracellular Fixation & Permeabilisation Buffer Set Intracellular Fixation & Permeabilisation Buffer Set Permeabilisation buffer Fixation buffer

8 protocols using fixation permeabilisation buffer

Phenotypic analysis of activated PBMCs

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Flow-cytometry analysis was performed on 52 frozen peripheral blood mononuclear cells (PBMCs) prior thawing. Egressed cells from one parotid MALT-L and PBMCs were stimulated with PMA (50 ng/mL), ionomycyn (750 ng/mL), Brefeldin-A (10 µg/mL) in RPMI complete (RPMI +10% fetal bovine serum) medium, for 3 hours. Cells were stained using Zombie Aqua Live/Dead kit (Biolegend) for 15 min, washed and incubated for 10 min with human Fc TruStain FcX (Biolegend). Cells were stained for surface antigens, fixed, permeabilised (fixation-permeabilisation buffer; eBioscience) and stained for intracellular cytokines. Antibodies used are listed in online supplementary table S2. Cells were acquired using a LSR Fortessa II (BD Biosciences) flow cytometer and analysed with FlowJo V.10 software.

Pontarini E., Murray-Brown W.J., Croia C., Lucchesi D., Conway J., Rivellese F., Fossati-Jimack L., Astorri E., Prediletto E., Corsiero E., Romana Delvecchio F., Coleby R., Gelbhardt E., Bono A., Baldini C., Puxeddu I., Ruscitti P., Giacomelli R., Barone F., Fisher B., Bowman S.J., Colafrancesco S., Priori R., Sutcliffe N., Challacombe S., Carlesso G., Tappuni A., Pitzalis C, & Bombardieri M. (2020). Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjögren’s syndrome with ectopic germinal centres and MALT lymphoma. Annals of the Rheumatic Diseases, 79(12), 1588-1599.

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Transcription Factor Analysis in Cells

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For analysis of transcription factors, cells were resuspended in 400 μl fixation/permeabilisation buffer (eBioscience) and incubated at 4 °C for between 1 and 18 h. Following incubation, cells were resuspended and washed twice in 1 ml permeabilisation buffer (eBioscience). In total 50 μl of antibody or isotype control (diluted in permeabilisation buffer) was added to each sample. Cells were resuspended by gentle vortex and incubated at room temperature for 30 min. Finally, cells were washed in 2 ml of FACS buffer and resuspended in 200 μl FACS buffer for acquisition.

Johnston C.J., Smyth D.J., Kodali R.B., White M.P., Harcus Y., Filbey K.J., Hewitson J.P., Hinck C.S., Ivens A., Kemter A.M., Kildemoes A.O., Le Bihan T., Soares D.C., Anderton S.M., Brenn T., Wigmore S.J., Woodcock H.V., Chambers R.C., Hinck A.P., McSorley H.J, & Maizels R.M. (2017). A structurally distinct TGF-β mimic from an intestinal helminth parasite potently induces regulatory T cells. Nature Communications, 8, 1741.

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Multiparametric Flow Cytometry Analysis of Immune Cells

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Thymic glands, spleens and inguinal lymph nodes (ILNs) were harvested from Hpa-tg and WT mice 7-days post CIA injection (n = 7). Single cell suspensions from these organs were prepared as described earlier31 (link) and stained with surface markers: CD4, CD8, CD25, CXCR5, PD-1, CD19, B220, Ly6G, CD11c, PDCA-1, CXCR6 and CD11b (Supplementary Table S2). The cells were then fixed and permeabilised with Fixation Permeabilisation buffer (eBioscience) for staining of intracellular markers: Foxp3, Helios, IFN-γ, Ki-67 and IL-17a (Supplementary Table S2). The samples were analysed on LSRFortesa (BD) using DivaDacker software (BD). One million events were counted for each sample and the fluorescence minus one and single stained controls were used for gating strategies as described earlier31 (link). The FCS files were analysed on FlowLogic software (Inivai Technologies).

Digre A., Singh K., Åbrink M., Reijmers R.M., Sandler S., Vlodavsky I, & Li J.P. (2017). Overexpression of heparanase enhances T lymphocyte activities and intensifies the inflammatory response in a model of murine rheumatoid arthritis. Scientific Reports, 7, 46229.

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Multiparametric Analysis of Macrophages

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Samples were blocked with 5 μg/mL anti CD16/32 (clone 2.4G2; produced in house) in ImageStream buffer (dPBS containing 1% FCS (Sigma), 2mM EDTA (Sigma)). Cells were stained for F4/80 PE (Clone: BM8 (BioLegend)) for 25 min at 4°C. Samples were washed in ImageStream buffer and fixed in 1% paraformaldehyde for 10 min at room temperature followed by permeabilization with Fixation/ Permeabilisation buffer (eBioscience) for 30 min at 4ºC and washed with 1× permeabilization buffer (eBioscience). Samples were stained with TOPRO3 (1:2000) for 10 min at room temperature and washed once with FACS buffer ready to acquire on the ImageStream. Data acquisition was performed on ImageStream^X (Amnis/EMD Millipore, Seattle, WA) equipped with 405, 488, 561, and 642 nm lasers. Single cells were discriminated from cell aggregates based on area and aspect ratio, in focus macrophages were selected based on high gradient RMS of the bright field image and F4/80 expression. The F4/80+ gate was drawn based on an FMO control and a minimum of 5000 F4/80+ cells were collected run at a low flow rate setting. Images of cells were acquired with a ×40 objective including bright field images (Channels 1 & 9; 430–480nm) F4/80 PE (Channel 3; 560–595nm), TOPRO3 (Channel 11; 660–740nm). All data analysis was performed using the IDEAS® software version 6.

Jackson‐Jones L.H., Rückerl D., Svedberg F., Duncan S., Maizels R.M., Sutherland T.E., Jenkins S.J., McSorley H.J., Bénézech C., MacDonald A.S, & Allen J.E. (2016). IL‐33 delivery induces serous cavity macrophage proliferation independent of interleukin‐4 receptor alpha. European Journal of Immunology, 46(10), 2311-2321.

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Characterization of Th17 and Treg Cells

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Treg cells were characterised by the expression of the specific transcription factor Foxp3 and surface activation marker CD25. After being labelled with FITC anti‐mouse CD4 and APC anti‐mouse CD25 antibodies (Biolegend), the cells were stained with fixation/permeabilisation buffer (eBioscience, California, USA) according to the manufacturer’s instructions and then labelled with a PE anti‐mouse Foxp3 antibody (Biolegend). For Th17 cell staining, cells were stimulated at 37°C for 5 h with a Cell Activation Cocktail containing Brefeldin A (Biolegend) and then labelled with a FITC anti‐mouse CD4 antibody. After being permeabilised with intracellular staining fixation/permeabilisation buffer (Biolegend) according to the manufacturer’s instructions, the cells were stained with a PE anti‐mouse IL‐17 antibody (Biolegend).⁵⁸ The stained cells were detected using an LSRFortessa cell analyser (BD, New York, USA). The data were analysed with FlowJo software, and the Th17 and Treg cell percentages among the total CD4⁺ T cells and the Th17/Treg ratio were statistically analysed.

Jia L., Wu R., Han N., Fu J., Luo Z., Guo L., Su Y., Du J, & Liu Y. (2020). Porphyromonas gingivalis and Lactobacillus rhamnosus GG regulate the Th17/Treg balance in colitis via TLR4 and TLR2. Clinical & Translational Immunology, 9(11), e1213.

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Multiparameter Flow Cytometry Panel

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Splenocytes were stained with fixable viability dye eFluor780 (1:1,000) before staining for surface antigens CD4-A700 (GK1.5, 1:100), CD69-PE-Cy7 (H1.2F3, 1:300), CD62L-PerCP-Cy5.5 (MEL-14, 1:200), LAG-3- PE (C9B7W, 1:200), TIM-3-PE (8B.2C12, 1:200) (all eBioscience) and PD-1-PE-Cy7 (29F.1A12, 1:300), TIGIT-APC (1G9, 1:200), CD49b-PE (HMα2, 1:100) (all Biolegend). For staining of transcription factors and Ki67, cells were then fixed with Fixation/permeabilisation buffer and antibodies were diluted in permeabilisation buffer (both eBioscience); Ki67-PE (SolA15, 1:200), c-Maf-eFluor660 (SYMOF1, 1:200), NFIL-3-PE (S2M-E19, 1:200), FoxP3-eFluor450 (FJK-16S, 1:100). Data were collected using an LSR II flow cytometer (BD Biosciences) and analysed using FlowJo software (Treestar).

Burton B.R., Britton G.J., Fang H., Verhagen J., Smithers B., Sabatos-Peyton C.A., Carney L.J., Gough J., Strobel S, & Wraith D.C. (2014). Sequential transcriptional changes dictate safe and effective antigen-specific immunotherapy. Nature Communications, 5, 4741.

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Flow Cytometric Analysis of Lung Immune Cells

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Lung single cell suspensions for flow cytometric analysis were prepared as previously described^{13 (link)}. Live cells were stained with fluorescently labelled antibodies against Siglec F, FcεRI, CD64, Ly-6G, CD11c, IFNGR1 and viability dye eFluor^™ 780 (ThermoFisher Scientific, reference: 65–0865–14). Antibodies used in this study are listed in Supplementary Table 2. After washing with FACS buffer (PBS containing 2% calf serum and 1 mM EDTA), cells were stained with streptavidin conjugated with PE-CF594 (1/300) for 15 mins at 4°C in the dark. Cells were subsequently fixed and permeabilized with 200 μl 1x Fixation/Permeabilisation Buffer (eBioscience, 00-5521-00) as per the manufacturer’s instructions, and then stained with a polyclonal FITC-anti-Legionella antibody (ViroStat, cat # 6053). Total numbers for each cell type were enumerated from the lungs by adding 2 × 10⁴ APC-labelled beads (BD Calibrite, reference: 340487) into each sample prior to flow cytometry analysis. Dead cells were detected and excluded based on eFluor 780 fluorescence. Data were analysed with FlowJo software.

Yang C., McDermot D.S., Pasricha S., Bedoui S., Lenz L.L., van Driel I.R, & Hartland E.L. (2019). Interferon γ receptor downregulation facilitates Legionella survival in alveolar macrophages. Journal of leukocyte biology, 107(2), 273-284.

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In Vivo Cell Proliferation in Pinnae

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Mice received 1 mg BrdU (Sigma-Aldrich) i.p. daily for the final 4 days before harvest of pinnae in order to determine in vivo cell proliferation. DEC were then recovered, and blocked with 1 μg anti-CD16/32 mAb in goat-serum. Subsequently, DEC were labelled for surface expression of CD3, CD4, MHC-II, F4/80 and CD11b (all eBioscience) in PBS supplemented with 1% FCS. Cells were then washed and incubated in 1x Fixation/Permeabilisation buffer (eBioscience) for 1 hour at 4°C before being washed and incubated for 1 hour at 37°C in 100 μg DNase (Sigma-Aldrich). Finally, cells were labelled for 45 minutes at room temperature with anti-BrdU APC mAb, or rat IgG1 APC (eBioscience), in 1x Permeabilisation buffer as per the manufacturer’s protocol.

Sanin D.E., Prendergast C.T., Bourke C.D, & Mountford A.P. (2015). Helminth Infection and Commensal Microbiota Drive Early IL-10 Production in the Skin by CD4+ T Cells That Are Functionally Suppressive. PLoS Pathogens, 11(5), e1004841.

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