Cytofix cytoperm fixation and permeabilization solution kit

Manufactured by BD

Sourced in United States

The Cytofix/Cytoperm fixation and permeabilization solution kit is a laboratory product designed for the preparation of cells for intracellular staining and flow cytometry analysis. The kit provides reagents for the fixation and permeabilization of cells, allowing for the detection of intracellular proteins and other cellular components.

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Lab products found in correlation

Perm wash buffer, by BD (1 mentions) Northern lights instrument, by Cytek (1 mentions) Fc receptor block, by Miltenyi Biotec (1 mentions) Brefeldin a, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Golgistop, by BD (1 mentions) Cyan flow cytometer, by Beckman Coulter (1 mentions) Live dead fixable green dead cell stain, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by Merck Group (1 mentions) Cyan adp analyzer, by Beckman Coulter (1 mentions) α cd4 clone gk1, by BioLegend (1 mentions) αcd8 clone 53 6.7, by BioLegend (1 mentions) αphospho mtor clone mrrby, by Thermo Fisher Scientific (1 mentions) αakt clone 55, by BD (1 mentions)

Close spelling variants of this product

BD Cytofix/Cytoperm plus fixation and permeabilization solution kit BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit BD Cytofix/Cytoperm fixation and permeabilization kit Cytofix/Cytoperm Fixation/Permeabilization solution BD Cytofix/Cytoperm Fixation/Permeabilization Kit BD Cytofix/Cytoperm™ Fixation/Permeabilization Cytofix Fixation and Permeabilization solution BD Cytofix/Cytoperm Permeabilization Solution Cytofix/Cytoperm Fixation solution

6 protocols using cytofix cytoperm fixation and permeabilization solution kit

Phospho-MET and Total MET Detection in Plasma Cells

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CD138 + plasma cells were isolated from bone marrow of MM patients. Cells were fixed and permeabilized with BD cytofix/cytoperm fixation and permeabilization solution kit (BD Biosciences, San Jose, CA). For detection of phospho-MET, cells were incubated with phospho-MET(Y1234/1235) for 30 minutes at room temperature followed by a wash with 1 × BD Perm/Wash Buffer. Control cells were incubated with normal rabbit IgG. Next, cells were incubated with secondary antibody (goat anti-rabbit IgG-phycoerythrin) for 30 minutes at 4°C. Cells were washed with 1 × BD Perm/Wash Buffer and analyzed by flow cytometry.
For detection of total MET, cells were incubated with anti-human HGF R/c-MET-fluorescein or normal mouse IgG1–fluorescein isothiocyanate (for control cells) for 30 minutes at room temperature, washed with 1 × BD Perm/Wash Buffer, and analyzed by flow cytometry. Antibody information is provided in Supplementary Table 1.

Zaman S., Shentu S., Yang J., He J., Orlowski R.Z., Stellrecht C.M, & Gandhi V. (2015). Targeting the Pro-Survival Protein MET with Tivantinib (ARQ 197) Inhibits Growth of Multiple Myeloma Cells. Neoplasia (New York, N.Y.), 17(3), 289-300.

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Quantifying SARS-CoV-2 Spike-Induced IFN-γ Secretion

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The levels of IFN-γ secretion by NK cells were measured by the following procedures. Around 1×10⁶ cells/well thawed PBMCs were plated in U-bottom 96 well plate containing complete RPMI-1640 medium (Gibco) with 5% heat inactivated FBS (Gibco). Cells were cultured in medium alone (unstimulated) or stimulated with SARS-CoV-2 spike peptide pools (S1 + S2, 2 µg/mL respectively, SinoBiological) for 24h at 37°C and 5% CO₂. After 24 hours of culture, Brefeldin A (1:1000, eBioscience) was added to the culture for an additional 4 hours. Next, cells were harvested and stained with a mixture of surface antibodies, including CD3-AF532, CD19-Super Bright 436, CD16-eFlour 450 and CD56-BV711. Fc-receptor block (Miltenyi Biotec) was added simultaneously to incubate in the dark at 4°C for 30 minutes. After the surface staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm Fixation and Permeabilization Solution Kit, and stained with intracellular antibody IFN-γ-PE-Cy7 for 30 minutes at room temperature in the dark. Stained cells were resuspended in FACS buffer and flow cytometry was performed using a Cytek™ Northern Lights instrument.

Cao C., Jiang J., Liu M., Dai Y., Chang T., Ji T, & Gong F. (2023). Longitudinal evaluation of innate immune responses to three doses of CoronaVac vaccine. Frontiers in Immunology, 14, 1277831.

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Th17 Cell Activation and Fixation

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The Th17 cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) and 1 µg/mL ionomycin (Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences) for 6 h at day 7. Cells were fixed with Cytofix/Cytoperm Fixation and Permeabilization Solution Kit (BD Biosciences).

Mutoh T., Shirai T., Ishii T., Shirota Y., Fujishima F., Takahashi F., Kakuta Y., Kanazawa Y., Masamune A., Saiki Y., Harigae H, & Fujii H. (2020). Identification of two major autoantigens negatively regulating endothelial activation in Takayasu arteritis. Nature Communications, 11, 1253.

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Caspase-3 Expression Quantification

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Cells were fixed with the Cytofix/Cytoperm fixation and permeabilization solution kit (BD Pharmingen), stained with FITC-conjugated anti-caspase-3 antibody (BD Pharmingen), and analyzed using CyAn flow cytometer (Beckman Coulter, Fullerton, CA). Data were evaluated using Flowjo (Tree Star Inc., Ashland, Oregon).

Zhang S., Yin Y., Li C., Zhao Y., Wang Q, & Zhang X. (2020). PAK4 suppresses TNF-induced release of endothelial microparticles in HUVECs cells. Aging (Albany NY), 12(13), 12740-12749.

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Functional T Cell Assay Protocol

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Functional T cell assays were performed as previously described (9 (link)). PBMCs were cultured for 5 days in the presence of 20 μg/ml peptides at a total cell concentration of 1 × 10⁶ cells/well in flat bottom 96-well plates in RPMI 1640 supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, and 10% pooled human serum. PBMCs were restimulated with peptides and anti-CD28 (BioLegend) on day 5 for 6 h with 5 μg/ml Brefeldin A (Sigma-Aldrich) being added for the last 4 h. Restimulated cells were treated with LIVE/DEAD^® Fixable Green Dead Cell Stain (Invitrogen) and thereafter stained for surface expression of CD3 (BioLegend), CD4, and CD14 (BD Biosciences). Cells were then stained intracellularly for IFN-γ and IL-17A (BioLegend) as well as CD154 (CD40L) (BD Biosciences) expression using the Cytofix/Cytoperm fixation and permeabilization solution kit (BD Biosciences). Samples were run on a CyAn™ ADP Analyzer (Beckman Coulter), and data were analyzed using FlowJo software, version 7.5.1 or higher (Tree Star). The gating strategy is depicted in Figure S2 in Supplementary Material. In some experiments, blocking antibodies for HLA-DR (clone L243), -DP (clone B7/21), and -DQ (clone SPLV3), all obtained from the Tetramer Core Facility (BRI, Seattle, WA, USA), were added to the cultures.

Gerstner C., Dubnovitsky A., Sandin C., Kozhukh G., Uchtenhagen H., James E.A., Rönnelid J., Ytterberg A.J., Pieper J., Reed E., Tandre K., Rieck M., Zubarev R.A., Rönnblom L., Sandalova T., Buckner J.H., Achour A, & Malmström V. (2016). Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted α-Enolase T Cell Epitope in Rheumatoid Arthritis. Frontiers in Immunology, 7, 494.

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Cell Surface and Intracellular Staining

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We performed cell surface and intracellular staining as previously described^{25 (link)}. We conducted cell surface staining using antibodies, including αCD4 (clone GK1.5; Biolegend, San Diego, CA, USA), αCD19 (clone 6D5; Biolegend), αCD43 (clone S11; Biolegend), αB220 (clone RA3-6B2; Biolegend), αCD8 (clone 53-6.7; Biolegend), αTLR2 (clone QA16A01; Biolegend), and αCD98 (clone RL388; Biolegend).
For intracellular staining, the cells were permeabilized using a Cytofix/Cytoperm fixation and permeabilization solution kit (BD Biosciences, Franklin Lakes, NJ, USA) and stained with antibodies, including α-phospho-mTOR (clone MRRBY; eBioscience, San Diego, CA, USA) and αAkt (clone 55; BD Biosciences).
We detected stained cells using flow cytometry (LSR II, BD Biosciences) and conducted data analysis with FlowJo software (version 10.6.2; Ashland, OR, USA).

Li Q., Wang J., Islam H., Kirschning C., Lu H., Hoffmann D., Dittmer U, & Lu M. (2021). Hepatitis B virus particles activate B cells through the TLR2–MyD88–mTOR axis. Cell Death & Disease, 12(1), 34.

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