Intracellular fixation buffer

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Intracellular fixation buffer is a solution used for the preservation and stabilization of cellular structures, particularly intracellular proteins, during sample preparation for various analytical techniques. It helps maintain the native conformation and distribution of intracellular components within cells.

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Intracellular Fixation & Permeabilization Buffer Set (135 citations) Fixation buffer (92 citations) Intracellular fixation and permeabilization buffer (23 citations) Intracellular Fixation & Permeabilization Buffer Set Kit (6 citations) Intracellular Fixation & Permeabilization Buffer kit (4 citations) Intracellular Fixation & Permeabilisation Buffer Set (3 citations) EBioscience™ Intracellular Fixation & Permeabilisation Buffer Set (3 citations) Intracellular Fixation & Permeabilization Buffer Set for cytokines (2 citations) Intracellular fixation & permeabiliztion buffer (2 citations) EBioscience intracellular fixation buffer (2 citations)

33 protocols using intracellular fixation buffer

Comprehensive Immune Profiling of Single-Cell Suspensions

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Surface staining of single cell suspensions was performed for 30 min at 4°C as previously described.²⁷ (link)^,²⁸ (link) Intracellular staining for FOXP3 and Ki67 was performed using the FOXP3 Transcription Factor Staining Buffer Set (eBioscience) per manufacturer’s protocol. Cytokine production was evaluated by intracellular staining after in vitro stimulation with Cell Stimulation Cocktail (eBioscience) for 4 h, using the intracellular fixation buffer (eBioscience), as previously described.²⁸ (link) CXCR3 staining in the tumor was also performed using the intracellular fixation buffer (eBioscience), given that it has been demonstrated in the literature that CXCR3 receptor can be internalized upon ligand binding.⁶² (link)^,⁶³ (link) Data acquisition was performed on Attune NxT cytometer (ThermoFisher) and Novocyte Quanteon (Agilent), and data analyzed by FlowJo software (TreeStar).

Lim R.J., Salehi-Rad R., Tran L.M., Oh M.S., Dumitras C., Crosson W.P., Li R., Patel T.S., Man S., Yean C.E., Abascal J., Huang Z., Ong S.L., Krysan K., Dubinett S.M, & Liu B. (2024). CXCL9/10-engineered dendritic cells promote T cell activation and enhance immune checkpoint blockade for lung cancer. Cell Reports Medicine, 5(4), 101479.

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Immune Cell Isolation and Characterization

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Lymphocytes were obtained from the blood, spleen, and lymph nodes. Erythrocytes were lysed. Single-cell suspensions were prepared in phosphate-buffered saline (PBS) and stained with fluorophore-conjugated antibodies. All antibodies used in this study are listed in Supplementary Table 1. For intracellular staining, single-cell suspensions were obtained from skin draining lymph nodes and cultured for 5 h with PMA (50 ng/mL) plus ionomycin (500 ng/mL). Brefeldin A (Thermo Fisher Scientific) was added during the final 4 h of incubation. After stimulation, the cells were washed with PBS and fixed with intracellular fixation buffer (Thermo Fisher Scientific) followed by cell permeabilization with permeabilization buffer (Thermo Fisher Scientific). The cells were stained with fluorescently conjugated anticytokine antibodies. Data were obtained using a Canto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo, LLC).

Kim S., Lee S.Y., Bae S., Lee J.K., Hwang K., Go H, & Lee C.W. (2020). Pellino1 promotes chronic inflammatory skin disease via keratinocyte hyperproliferation and induction of the T helper 17 response. Experimental & Molecular Medicine, 52(9), 1537-1549.

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Multiparametric Flow Cytometry for Immune Cells

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Single-cell suspensions were washed in PBS, and Live/Dead staining (Thermo Fisher Scientific) was performed. Samples were Fc-blocked using anti-CD16/32 (2.4G2, RRID:AB_394656) (BD Biosciences) and mouse serum (Bio-Rad). Blocking and subsequent surface staining was performed using PBS containing 2 mM EDTA, 2% FBS, and 0.05% NaN₃. Abs used for staining are listed in Table I. Following surface staining, cells were incubated with intracellular fixation buffer (Thermo Fisher Scientific) prior to permeabilization for intracellular staining. For secondary detection of Ym1 and RELM-α, Zenon goat (RRID:AB_2753200) and rabbit (RRID:AB_2572214) Ab labels (Thermo Fisher Scientific) were used. For Ym1, RELM-α, and pro–IL-1β intracellular staining, cells were directly stained without stimulation or protein transport inhibition. For cell quantification, some samples were spiked with 10 μm polystyrene beads (Sigma-Aldrich) prior to acquisition. Data were acquired on a BD LSRFortessa flow cytometer and analyzed using FlowJo v10 software.

Chenery A.L., Alhallaf R., Agha Z., Ajendra J., Parkinson J.E., Cooper M.M., Chan B.H., Eichenberger R.M., Dent L.A., Robertson A.A., Kupz A., Brough D., Loukas A., Sutherland T.E., Allen J.E, & Giacomin P.R. (2019). Inflammasome-Independent Role for NLRP3 in Controlling Innate Antihelminth Immunity and Tissue Repair in the Lung. The Journal of Immunology Author Choice, 203(10), 2724-2734.

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Flow Cytometry Cell Staining Protocol

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Cells were washed with PBS, pelleted by centrifugation (400 × g for 5 min at 4°C) and stained with Zombie NIR Fixable Dye for 30 min in the dark and on ice. After washing with PBS, cells were incubated with 50 μl of FACS buffer (2% FBS in PBS) mixed with anti-CD16/CD32 monoclonal Abs. Cells were then stained with the fluorochrome-labeled Abs for 15 min in the dark and on ice. Cells were washed twice and resuspended in 100 μl of FACS buffer and fixed using 100 μl Intracellular Fixation Buffer (Thermo Fisher Scientific). For intracellular staining, cells were permeabilized and stained using 200 μl 1× Permeabilization Buffer prepared from 10× Permeabilization Buffer (Thermo Fisher Scientific). Cells were washed twice using 1× Permeabilization Buffer and resuspended in 100 μl of FACS buffer. All compensations were set up using OneComp beads (eBioscience), or if not applicable, cells were stained with a dye. Samples were acquired on BD FACSCanto II or Attune NxT Acoustic Focusing Cytometer using FACSDiva (BD Biosciences) or Attune NxT software (Thermo Fisher Scientific), and the data were analyzed using FlowJo software (Tree Star).

Oleszycka E., Rodgers A.M., Xu L, & Moynagh P.N. (2021). Dendritic Cell–Specific Role for Pellino2 as a Mediator of TLR9 Signaling Pathway. The Journal of Immunology Author Choice, 207(9), 2325-2336.

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Flow Cytometric Analysis of LBR Expression

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After incubation in Intracellular Fixation Buffer (Thermo Fischer Scientific, Waltham, MA) for 30 min at room temperature and permeabilization with cold 100% methanol on ice for 30 min, cells were stained with FITC-conjugated anti-LBR mAb (clone e398L, Abcam, Cambridge, MA) diluted 1/50 in blocking buffer (1% FBS in PBS). After washing, cells were resuspended in PBS and 10,000 events/sample were acquired using an Accuri C6 Cytometer (BD Biosciences, East Rutherford, NJ) followed by FlowJo V10 analysis.

Silva-Del Toro S.L, & Allen L.A. (2021). Microtubules and Dynein Regulate Human Neutrophil Nuclear Volume and Hypersegmentation During H. pylori Infection. Frontiers in Immunology, 12, 653100.

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Isolation and Analysis of Non-Epithelial Intestinal Cells

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Non-epithelial cells were prepared from the lamina propria of the small intestine as described previously.^{71 (link)} After epithelium was removed by incubation in 2 mM EDTA, the remaining intestinal tissue was chopped into small pieces, and digested with collagenase (0.5 mg/ml, Wako). The obtained cell suspension was suspended in 40% Percoll solution (GE Healthcare) and loaded on a 80% Percoll solution. After centrifugation for 20 min at 880 × g at room temperature, cells at interface between the two Percoll solutions were harvested. After stimulation for 4 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of GolgiPlug (1:1000 dilution), cell surface was stained with FVD506 (eBioscience 65-0866-14), APC/Cy7-labeled anti-CD45.2 antibody (BioLegend 109824, clone 104), APC-labeled anti-CD4 antibody (TONBO 20-0042, clone RM4-5), PE/Cy7-labeled TCRβ antibody (TONBO 60-5961, clone H57-597). The cells were fixed with Intracellular Fixation Buffer (eBioscience 00-8222) and then perforated in Permeabilization Buffer (eBioscience 00-8333). Intracellular cytokines were stained with PE-labeled anti-IL-17A (BioLegend 506904, clone TC11-18H10) and FITC-labeled anti-IFN-γ (BioLegend 505806, clone XMG1.2) in Permeabilization Buffer, and analyzed using LSR Fortessa^TM X-20 Cell Analyzer (BD Bioscience) and FlowJo (BD Bioscience).

Yamazaki S., Inohara N., Ohmuraya M., Tsuneoka Y., Yagita H., Katagiri T., Nishina T., Mikami T., Funato H., Araki K, & Nakano H. (2022). IκBζ controls IL-17-triggered gene expression program in intestinal epithelial cells that restricts colonization of SFB and prevents Th17-associated pathologies. Mucosal Immunology, 15(6), 1321-1337.

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PBMC Cytokine Induction Assay

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3–5 × 10⁵ PBMCs were seeded in 96-well plates and stimulated with MEDI9197 (in DMSO), CpG (ODN 2395 VacciGrade™ in H₂O, Invivogen), STING agonist (2′3’-c-di-AM (PS)2 (Rp,Rp) VacciGrade™ in H₂O, Invivogen), Resiquimod (in DMSO, Sigma-Aldrich), or Imiquimod (in DMSO, R&D Systems) at the indicated concentrations for 24 h at 37 °C. Flow cytometry was performed by standard procedures with intracellular fixation buffer from eBioscience. See Additional file 1 for a list of Flow cytometry antibodies. IFN-gamma was analysed by ELISA (Standard: human recombinant IFNγ (R & D). Capture antibody: Anti-human IFNγ (Clone NIB42, Pharmingen). Detection antibody: Biotinylated anti-human IFNγ (Clone 4S.B3, Pharmingen) or MSD (Mesoscale Diagnostics). IFN-alpha was analysed by ELISA (MABTECH) and IL-12p70 by MSD (Mesoscale Diagnostics). For ELISAs, DELFIA readout (Perkin Elmer) was used.

Mullins S.R., Vasilakos J.P., Deschler K., Grigsby I., Gillis P., John J., Elder M.J., Swales J., Timosenko E., Cooper Z., Dovedi S.J., Leishman A.J., Luheshi N., Elvecrog J., Tilahun A., Goodwin R., Herbst R., Tomai M.A, & Wilkinson R.W. (2019). Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies. Journal for Immunotherapy of Cancer, 7, 244.

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Quantification of Murine CD4+ Treg Cells

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The isolated liver CD4+ T cells from individual mice were stained with FITC-labeled anti-CD4 (clone GK1.5, Ebioscience, USA) or APC-labeled anti-CD25 (clone PC61.5, Ebioscience, USA) for 30 minutes in the dark. After being washed, the cells were fixed with Intracellular Fixation buffer (Ebioscience, USA) and permeabilized with Foxp3/Transcription Factor Staining Buffer (Ebioscience, USA), followed by intracellularly staining with PE-labeled anti-FOXP3 (Ebioscience, USA). After being washed, the percentages of CD4+CD25+FOXP3+ Tregs in total CD4+ T cells were analyzed by flow cytometry in a flow cytometry machine (ACEA NovoCyte, USA or Beckman, USA). The data were analyzed by NovoExpressTM software or FlowJo software.

Li X., Zhu Z., Xia Z, & Xu B. (2022). FGF15 Protects Septic Mice by Inhibiting Inflammation and Modulating Treg Responses. Journal of Inflammation Research, 15, 6187-6197.

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Intracellular FACS Analysis of Cell Cycle

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MEFs were isolated and cultured as previously described (Li et al., 2011 (link)). MEFs were treated with either control vehicles or designated cell cycle inhibitors, then digested and collected as single cell suspensions. The cell suspension was washed with PBS and then fixed with intracellular fixation buffer (eBiosciences). For intracellular FACS analyses, cells were permeabilized with permeabilization buffer (eBiosciences) and then incubated with GFP antibodies (see Supplementary Table 1) for 2 h at room temperature, followed by incubation with secondary antibodies (Alexa fluor, Life Technologies) for 1 h at room temperature. Samples were run and analyzed using a BD FACS Canto II instrument and software (BD Biosciences).

Jang J., Engleka K.A., Liu F., Li L., Song G., Epstein J.A, & Li D. (2020). An Engineered Mouse to Identify Proliferating Cells and Their Derivatives. Frontiers in Cell and Developmental Biology, 8, 388.

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Obtaining Immune Cell Subsets from Murine Spleens

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Single cell suspensions were prepared by mechanical maceration from spleens of saline and AVP-infused dams followed by density gradient separation (FicoLite LM, Atlanta Biologicals, Lawrenceville, GA) to obtain MNCs. Cell suspensions were then stained with fluorochrome-conjugated or biotinylated antibodies (Supplemental Table 1), followed by streptavidin-conjugated fluorochromes. Fluorochrome-conjugated, purified rat immunoglobulins were used as isotype controls for background fluorescence. All cell samples were incubated with anti-CD16/32 (clone 2.4G2) and rat serum during staining to prevent background FcγR binding. Intracellular Fixation and Permeabilization Buffer Set (eBioscience, San Diego, CA) was used per protocol for intracellular staining (ICS) of cytokines. Following staining, cells were fixed with either 0.1% formaldehyde or intracellular fixation buffer (eBioscience, San Diego, CA) where appropriate. Flow cytometric data were obtained within 24 hours using a Becton Dickinson LSR II (San Jose, CA) and analyzed using FlowJo software (Treestar Inc., Ashland, OR). Dead cells were excluded by forward/orthogonal light scatter characteristics. Single cells were identified via forward scatter-area (FSC-A) versus side scatter-width (SSC-W). Gating strategies of CD4+ T cells and dendritic cells (DCs) are shown in Supplemental Figure 2.

Scroggins S.M., Santillan D.A., Lund J.M., Sandgren J.A., Krotz L.K., Hamilton W.S., Devor E.J., Davis H.A., Pierce G.L., Gibson-Corley K.N., Sigmund C.D., Grobe J.L, & Santillan M.K. (2018). Elevated Vasopressin in Pregnant Mice Induces T Helper Subset Alterations Consistent with Human Preeclampsia. Clinical science (London, England : 1979), 132(3), 419-436.

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