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Cytofix perm

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CytoFix/Perm is a laboratory reagent used for fixation and permeabilization of cells. It is designed to prepare cells for subsequent intracellular staining and flow cytometry analysis.

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Lab products found in correlation

Penicillin, by Thermo Fisher Scientific (2 mentions) Ionomycin, by Merck Group (2 mentions) Brefeldin a, by Merck Group (2 mentions) Facscanto 2, by BD (2 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Facsaria 3 cell sorter, by BD (1 mentions) Facsdiva software, by BD (1 mentions) Foxp3 fix perm buffer, by BioLegend (1 mentions) Pe anti ki67, by BioLegend (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Surface antibodies, by Thermo Fisher Scientific (1 mentions) Live dead fixable aqua dye, by Thermo Fisher Scientific (1 mentions) Foxp3 pe, by BD (1 mentions) Il 17a percp cy5, by BD (1 mentions) Rorγt pe, by BD (1 mentions) Goat anti mouse igg pe, by BD (1 mentions) Ab24011, by Abcam (1 mentions) Nucleofector 2b, by Lonza (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Cytofix (417 citations) Cytofix/perm kit (20 citations) Cytofix/perm buffer (16 citations) Cytofix/Cytoperm and Perm/Wash Buffer (7 citations) BD Cytofix and Phosflow Perm/Wash buffers (5 citations) Cytofix/Phosflow perm Buffer III (4 citations) BD CytoFix/Perm reagent (3 citations) Cytofix/perm solution (3 citations) BD Perm/Wash and Cytofix/Cytoperm buffers (2 citations) BD Cytofix Buffer and Perm/Wash reagent (2 citations)

12 protocols using cytofix perm

1

Multiparametric Flow Cytometry Analysis

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The following anti-mouse antibodies were used for detection and enrichment of immune cells: APC anti-CD3 (17A2), PE anti-TCRβ (H57–597), APC-Cy7 anti-CD4 (GK1.5), Alexa Fluor® 488 anti-Foxp3 (R16–715), and PE-Cy7 anti-CD8a (53–6.7) (BD Pharmingen); PE anti-CTLA-4 (UC10–4B9) and PE anti-ICOS (7E.17G9) (eBioscience). PE anti-Ki67 (Biolegend), Anti-EGFR antibody (ab30) (Abcam). For cell surface staining, cells were incubated with corresponding antibodies in PBS for 15 min on ice before analysis on a FACSAria™ III cell sorter (FACSDiva software, BD). Dead cells that stained by propidium iodide (2 μg/ml) were excluded. For intracellular cytokine staining, cells were fixed and permeabilized with BD Cytofix/perm and Perm/wash buffer according to the manufacturer's protocol. Then cells were stained at room temperature for 30 min with Alexa Fluor® 488 anti-IFN-γ (XMG1.2, Biolegend), Alexa Fluor® 647 anti-TNF-α (MP6-XT22, Biolegend), APC anti-perforin (eBioOMAK-D, eBioscience) and PE anti-granzyme B (NGZB, eBioscience) respectively before analysis on a BD LSRII flow cytometer. For Foxp3 staining, Foxp3 fix/perm buffer (Biolegend) set was used according to the manufacturer's instruction. All flow cytometry data was analyzed with Flowjo 7.6.1 software. Cell sorting was performed on a FACSAria™ III cell sorter (FACSDiva software, BD) based on cell surface marker staining.
Yuan C.H., Sun X.M., Zhu C.L., Liu S.P., Wu L., Chen H., Feng M.H., Wu K, & Wang F.B. (2015). Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells. Oncotarget, 6(31), 32138-32153.
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2

Flow Cytometric Analysis of Immune Cells

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Surface staining and intracellular cytokine staining (ICS) were described previously [42 (link)]. Briefly, single-cell suspensions from peripheral blood, spleen or liver were stained with LIVE/DEAD Fixable Aqua Dye (Invitrogen) to exclude dead cells, then incubated with surface antibodies (eBiosciences) in combination with tetramers (NIH Tetramer Core Facility, Atlanta, GA). For ICS, cells were first cultured at 37°C for 5 hours in the complete medium (RPMI 1640 containing 10% FBS, 1% HEPES, 1% non-essential amino acid, 2 mM L-glutamine, 50 μM β-mercaptoethanol, 100 U/ mL penicillin and 100 μg/mL streptomycin; Gibco), in the presence of LLO190-201, GP33-41 or OVA257-264 peptides (1 μg/mL), or PMA (50 ng/mL) + Ionomycin (0.5 μg/mL) (Sigma Aldrich). Brefeldin A and monensin were added to the culture for the last 4 hours. Cells were stained for viability and surface antigens as described above, followed by permeabilization (BD Cytofix/Perm) for intracellular staining. Samples were acquired on LSRII flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (Treestar). Details about the reagents and softwares we used are shown in S1 Table.
Yang C., Liu Z., Yang Y., Cocka L.J., Li Y., Zeng W, & Shen H. (2024). Chronic viral infection impairs immune memory to a different pathogen. PLOS Pathogens, 20(3), e1012113.
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3

Cytokine Production Assay for Innate Lymphoid Cells

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To examine ILC2 effector cytokine production, single cell suspensions of E-WAT or iWAT SVF were stimulated for 4 hours ex vivo with PMA (100 ng/mL) and ionomycin (1 ng/mL) in the presence of Brefeldin A (10 μg/mL) (all from Sigma-Aldrich) in a 37°C incubator (5% CO2). Cells were then surface stained, fixed and permeabilized using Cyto Fix/Perm (BD Pharmingen) according to manufacturer’s instructions before intracellular staining for IL-5 (APC-IL-5, clone TRFK5, eBioscience) and IL-13 (PE-IL-13, eBio13A, eBioscience). Monensin (1:1500) was also used for intracellular staining with rabbit anti-mouse MetEnk (bs-1759R, Bioss USA, Woburn, MA) or rabbit anti-mouse IgG (Isotype control, Bioss USA) followed by staining with goat anti-rabbit PE (sc-3739, Santa Cruz Biotechnology, Dallas, TX)
Brestoff J.R., Kim B.S., Saenz S.A., Stine R.R., Monticelli L.A., Sonnenberg G.F., Thome J.J., Farber D.L., Lutfy K., Seale P, & Artis D. (2014). Group 2 innate lymphoid cells promote beiging of adipose and limit obesity. Nature, 519(7542), 242-246.
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4

Multiparametric Flow Cytometry Analysis

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Flow cytometry Abs were purchased from BD Pharmingen, e-Bioscience and Biolegend (San Diego, CA). Single-cell suspensions, after anti-CD16/CD32 blocking, were stained for 25 min at 4°C with the following primary Abs or isotype controls: IgA-FITC (Clone C10-3), B220-PE (Clone RA3-6B2), CD138-APC (clone 281-2), CD5-PE (clone 53-7.3), CXCR5- PE-Cy7 (clone L138D7), PD1-FITC (clone J43), CD11b−APC (Clone M1/70), CD86-PE (Clone GL1), CD80-APC (Clone 16-10A1), CD11c-FITC (Clone HL3,), MHC-II-FITC (Clone 39-10-8), CD64-PerCP-Cy5.5 (clone X54-5/7.1), CD103-PE (Clone M290), CD19-PerCP (Clone 6D5), CD45-APC-Cy7 (Clone 30-F11), CD3-FITC (Clone 17A2), CD4-PE-Cy7 (Clone 4-5), TCRβ-PE (Clone H57-597), TCRδ-APC (Clone GL3). For intracellular IL-17A and FoxP3 staining, cells were fixed with Fix/Perm reagents (Cyto Fix/Perm, BD) for 30 min at 4°C and stained with IL-17A-PerCP-Cy5.5 (Clone TC11-18H10), RORγt-PE (Clone B2D) or FoxP3-PE (Clone MF23). Events were acquired on FACSCanto II (BD Biosciences) and were analyzed with Flowjo software version 7.6.1.
Zhao H., Yang J., Qian Q., Wu M., Li M, & Xu W. (2018). Mesenteric CD103+DCs Initiate Switched Coxsackievirus B3 VP1-Specific IgA Response to Intranasal Chitosan-DNA Vaccine Through Secreting BAFF/IL-6 and Promoting Th17/Tfh Differentiation. Frontiers in Immunology, 9, 2986.
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5

Quantification of RSV Entry and Binding

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PBMCs or isolated monocytes were treated with CytoD (1 μg/ml), Wisko (10 μM) or CPZ (5 μg/ml) for 30 min at 37 °C. For quantification of cell entry, monocytes were permeabilized with CytoFix/Perm (BD Biosciences) after incubation with RSV at MOI 1 (5 x 105 IU/well) for 1 h at 37 °C. Cells were incubated for 30 min on ice with mouse anti-respiratory syncytial virus fusion protein antibody (Ab24011; Abcam). Goat anti-mouse IgG PE (BD Pharmingen) was used as secondary antibody. For the quantification of RSV binding, co-incubations of PBMCs with RSV were performed for 1 h at 4 °C and cells were fixed with 1 % paraformaldehyde before staining.
Jans J., elMoussaoui H., de Groot R., de Jonge M.I, & Ferwerda G. (2016). Actin- and clathrin-dependent mechanisms regulate interferon gamma release after stimulation of human immune cells with respiratory syncytial virus. Virology Journal, 13, 52.
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6

Flow Cytometry Analysis of Viral Antigens

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Human skeletal muscle cells (HskMCs, Lonza, Cat #: CC-2561) were cultured in M199 (Life Technologies) supplemented with GlutaMax (Life Technologies), streptomycin, penicillin (Gibco), and 20% heat inactivated FBS (VWR) at 37 °C with 5% CO2. The cells were harvested by trypsinization, washed with PBS, and electroporated using human primary muscle cell transfection kit on Nucleofector 2b (Lonza) with 12 mg of mRNA per 106 cells following manufacturer’s electroporation program D-033. Post 24 hr harvested cells were fixed, permeabilized with CytoFix/Perm (BD) and stained with Cal09 HA (Immune Tech), Sing16 HA (30-2F11-F7-A5, GeneTex), Mich15 NA (6G6, Immune Tech) and Sing16 NA (40017-RP01, Sino Biologicals) specific Ab followed by PE conjugated goat anti-mouse IgG secondary Ab (Southern Biotech) or AF647 conjugated goat anti-rabbit IgG (Life Technologies). Then the Ab labeled cells were acquired by Fortessa (BD) and the expression of each protein was analyzed by FlowJo 10.6.2 (BD, Ashland, OR). Blots for each antigen were processed in the same experiment and were processed in parallel.
Chivukula S., Plitnik T., Tibbitts T., Karve S., Dias A., Zhang D., Goldman R., Gopani H., Khanmohammed A., Sarode A., Cooper D., Yoon H., Kim Y., Yan Y., Mundle S.T., Groppo R., Beauvais A., Zhang J., Anosova N.G., Lai C., Li L., Ulinski G., Piepenhagen P., DiNapoli J., Kalnin K.V., Landolfi V., Swearingen R., Fu T.M., DeRosa F, & Casimiro D. (2021). Development of multivalent mRNA vaccine candidates for seasonal or pandemic influenza. NPJ Vaccines, 6, 153.
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7

Intracellular Cytokine Profiling in Lymphocytes

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To detect intracellular cytokine production, 5 × 106 splenocytes or pulmonary lymphocytes were incubated at 37°C with 10 μM mixed peptides plus 1 μg/ml anti-CD28 mAb for 10 h, and treated with 10 μg/ml Brefeldin A (BD Pharmingen) for an additional 5 h. 1 × 106 cells were first stained with surface markers of PE-Cy7-CD4 or PerCP-CD8(BD Pharmingen) for 30 min, then fixed and permeabilized by Cyto Fix/Perm (BD Pharmingen) for 30 min. Intracellular staining was performed using FITC-IFN-γ, PE-IL-2, APC-TNF-α Abs (BD Biosciences). Flow cytometric data were acquired using a FACSCanto II (BD Biosciences) and analyzed using FlowJo software (TreeStar, San Carlos, CA).
Wu M., Zhao H., Li M., Yue Y., Xiong S, & Xu W. (2017). Intranasal Vaccination with Mannosylated Chitosan Formulated DNA Vaccine Enables Robust IgA and Cellular Response Induction in the Lungs of Mice and Improves Protection against Pulmonary Mycobacterial Challenge. Frontiers in Cellular and Infection Microbiology, 7, 445.
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8

Characterization of Myeloid-Derived Cells

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MDM were detached after 48 hrs treatment with PBA, by incubating in Acutase (Invitrogen) at 37°C for 30min, washed twice with PBS, and suspended in PBS/5% FCS. Cells were incubated with either monoclonal mouse anti-human CD35/FITC, CD11c/PE, CD16/PE.Cy5, CD11b/APC conjugated antibodies or respective IgG-conjugated antibodies (BD Bioscience) in the dark at 4°C for 30 min. Cells were washed, resuspended in CytoFix/Perm (BD Bioscience) for 20 min at 4°C and then washed twice and resuspended in PBS/5% FCS. An aliquot of unfixed cells were also incubated with propidium iodide. Ten thousand events were analysed for each sample by immunofluorescence using flow cytometry (BD FACSCalibur). Results were analysed using Flow Jo (version 10).
Coussens A.K., Wilkinson R.J, & Martineau A.R. (2015). Phenylbutyrate Is Bacteriostatic against Mycobacterium tuberculosis and Regulates the Macrophage Response to Infection, Synergistically with 25-Hydroxy-Vitamin D₃. PLoS Pathogens, 11(7), e1005007.
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9

Quantifying Mouse T Cell Subsets and IFNγ Production

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The antibodies were purchased from eBioscience, BD Pharmingen, or BioLegend and are as follows: rat anti-mouse CD8α (clone 53.6.7), rat anti-mouse CD4 (clone GK1.5), biotinylated anti-mouse CCR5 (clone HM-CCR5), anti-mouse CD3e (clone 145.2C11), anti-mouse CD45.2 (clone A20), rat IgG2b, rat IgG2c, and Hamster IgG-biotin isotype control. Additional CCR5 antibody, MC-68, was a generous gift from Dr. Matthias Mack (17 (link)). Between 2 x 105 and 1x106 cells were washed in ice-cold FACS buffer (PBS/2.5mM EDTA/0.1% BSA), followed by incubation in blocking buffer (10% normal mouse serum in FACS buffer) for 15 min at 4°C. Antibodies were added 30 minutes on ice, except for anti-CCR5 antibody, which was incubated for 1 h on ice. The samples were then washed twice with ice-cold FACS buffer and the samples were run on Accuri C6 or FACScalibur flow cytometer. Fold increase of CCR5 expression was determined by using the following formula: [(%CCR5+ experiment – %CCR5+ isotype control)/(%CCR5+ control – %CCR5+ isotype control)]. To detect IFNγ-producing T cells, after stimulation with anti-CD3, T cells were first stained with anti-CD8 and CD45.2 prior to fixing with CytoFix/Perm (BD). Cells were then washed with Perm Wash buffer (BD) and then stained with anti-IFNγ Ab (clone XMG1.2). The data were then analyzed using FlowJo software (FlowJo, Inc).
Askew D., Su C.A., Barkauskas D.S., Dorand R.D., Myers J., Liou R., Nthale J, & Huang A.Y. (2016). Transient surface CCR5 expression by naïve CD8+ T cells within inflamed lymph nodes is dependent on HEV interaction and augments helper T-cell dependent memory response. Journal of immunology (Baltimore, Md. : 1950), 196(9), 3653-3664.
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10

Intracellular Staining of NOX4 in SSc IgG-Treated HPASMCs

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For intracellular staining assay, HPASMC were treated with SSc IgG or N IgG (200 µg/ml) and PDGF (15 ng/ml) for 24 h and then permeabilized with Cyto Fix/Perm (Becton Dickinson). Cells were detached by trypsin, washed with PBS, and incubated with antibody against NOX4 (AbCam) for 24 h at 37°C. Cells were then washed and incubated with FITC-conjugated secondary antibody and resuspended in 1 ml PBS. For each sample, 10,000 events were collected. Data analysis was carried out using the WinMDI software.
Svegliati S., Amico D., Spadoni T., Fischetti C., Finke D., Moroncini G., Paolini C., Tonnini C., Grieco A., Rovinelli M., Funaro A, & Gabrielli A. (2017). Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro. Frontiers in Immunology, 8, 75.
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