Cytofix cytoperm fixation kit

Manufactured by BD

Sourced in Canada, Japan

The Cytofix/Cytoperm fixation kit is a laboratory product designed for cell fixation and permeabilization. It provides a solution for preparing cells for intracellular staining and flow cytometry analysis.

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Lab products found in correlation

Kc57 rd1 antibody, by Beckman Coulter (1 mentions) Golgistop protein transport inhibitor, by BD (1 mentions) Anti rankl ab, by R&D Systems (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Bz x710 all in one fluorescence microscope, by Keyence (1 mentions) Live dead fixable yellow dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Lsrfortessa x 20, by BD (1 mentions) Efluor780 fixable viability dye, by Thermo Fisher Scientific (1 mentions) Cd27 m t271, by BioLegend (1 mentions) Vascular endothelial growth factor (vegf), by Miltenyi Biotec (1 mentions) Cd16 fitc, by Miltenyi Biotec (1 mentions) Cd56 apc, by Miltenyi Biotec (1 mentions) Sdf 1, by Miltenyi Biotec (1 mentions) Cd3 percp, by Miltenyi Biotec (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Ifn γ, by Miltenyi Biotec (1 mentions) Monensin, by BD (1 mentions)

Close spelling variants of this product

BD Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit BD Cytofix/Cytoperm Fixation/Permeabilization Kit Cytofix/Cytoperm™ Fixation/Permeabilisation Solution Kit BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit Cytofix/Cytoperm fixation and permeabilization solution kit BD Cytofix/Cytoperm Fixation/Permeablization Kit BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with GolgiPlug™( BD Cytofix/Cytoperm fixation and permeabilization kit BD Cytofix/Cytoperm plus fixation and permeabilization solution kit

5 protocols using cytofix cytoperm fixation kit

Latently Infected Cell Activation Assay

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J-Lat 9.2 or 10.6 cells were seeded in 96 well plates at 200,000 cells/well and co-treated with a pro-inflammatory cytokine (10 ng/mL TNFα) or an LRA (10 μM prostratin, 0.1 μM panobinostat, 10 μM JQ1, or 10 mM HMBA) in the presence of test agents at defined concentrations or 0.1% DMSO vehicle control for 24 hours. Cells were then examined for GFP expression by flow cytometry. In a subset of experiments, cells were fixed and permeabilized using the Cytofix/Cytoperm fixation kit (BD Biosciences; Mississauga, ON, Canada), stained for intracellular p24^Gag (KC57-RD1 antibody, Beckman Coulter; Mississauga, ON, Canada), and examined for p24^Gag expression by flow cytometry.
OM-10.1 cells were seeded as above and cotreated with 1 ng/mL TNFα and test agents or 0.1% DMSO control for 24 hours. Cells were fixed, permeabilized, stained for p24^Gag as above, and examined for p24^Gag expression by flow cytometry.

Schonhofer C., Yi J., Sciorillo A., Andrae-Marobela K., Cochrane A., Harris M., Brumme Z.L., Brockman M.A., Mounzer K., Hart C., Gyampoh K., Yuan Z., Montaner L.J, & Tietjen I. (2021). Flavonoid-based inhibition of cyclin-dependent kinase 9 without concomitant inhibition of histone deacetylases durably reinforces HIV latency. Biochemical pharmacology, 186, 114462.

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Inflammatory Cytokine Production in Monocytes

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OCLs and pre-OCLs induced from peripheral monocytes were stimulated using 1 ng ml⁻¹ lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA), 5 μg ml⁻¹ anti-RANKL Ab (R&D Systems) or 5 μg ml⁻¹ anti-Mouse IgG2B κ isotype control Ab (BD BioSciences, San Diego, CA, USA), with GolgiStop protein transport inhibitor (BD BioSciences), and incubated for 5 h at 37°C. Cells were then stained for 15 min at 4°C with fluorescent antibody RANK-Alexa Fluor (AF)-488 (Novus, Littleton, CO, USA). Next, cells were fixed for 30 min at 26°C using Cytofix/Cytoperm™ fixation kit (BD) and stained for 30 min at 4°C with fluorescent antibodies: IL-8-PE (BioLegend), PE Mouse IgG2b κ isotype control (BD BioSciences) and cathepsin K-PE (Abcam, Tokyo, Japan). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Finally, cells were photographed using a BZ-X710-All-in-One Fluorescence Microscope (Keyence, Osaka, Japan).

Morita T., Shima Y., Fujimoto K., Tsuboi H., Saeki Y., Narazaki M., Ogata A, & Kumanogoh A. (2019). Anti-receptor activator of nuclear factor κB ligand antibody treatment increases osteoclastogenesis-promoting IL-8 in patients with rheumatoid arthritis. International Immunology, 31(5), 277-285.

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Single-cell immunophenotyping protocol

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Single-cell suspensions were stained for 30 minutes at 4^°C with different combinations of antibodies listed in the key resources table. Homemade 1G12 antibodies were used to stain for the 3A9 TCR.²³ (link)^,⁶⁶ (link) Biotin-labelled antibodies were revealed with fluorescently-coupled streptavidin. Dead cells were stained using LIVE/DEAD^TM Fixable Yellow Dead Cell Stain Kit (Thermo Fisher Scientific). For caspase staining, cells were treated with Cytofix/Cytoperm Fixation kit (BD Biosciences) as directed by the manufacturer. The FOXP3 Transcription Factor Staining Buffer Set (eBioscience) was used for transcription factor staining as directed by the manufacturer. Data were collected on an LSRFortessaX20 (BD) and analyzed with FlowJo software (BD Biosciences). B cells were gated as CD19⁺B220⁺ cells. TECs, cDC1, cDC2 and pDC were gated as EpCAM⁺CD45^-, CD11c^hiCD8α⁺CD11b^-, CD11c^hiCD8^-CD11b⁺, and CD11c^lowB220⁺, respectively. CD4SP, CD8SP and post-selection DP were gated as TCRβ⁺CD5⁺CD4⁺CD8^-, TCRβ⁺CD5⁺CD4^-CD8⁺, and TCβ⁺CD5⁺CD4⁺CD8⁺, respectively.

Lombard-Vadnais F., Chabot-Roy G., Zahn A., Rodriguez Torres S., Di Noia J.M., Melichar H.J, & Lesage S. (2022). Activation-induced cytidine deaminase expression by thymic B cells promotes T-cell tolerance and limits autoimmunity. iScience, 26(1), 105852.

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Characterizing CAR T Cell Phenotype

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Mouse anti-human antibodies to CD3 (OKT3), CD4 (OKT4; RPA-T4), CD8 (RPA-T8), CD45RA (HI100), CD27 (M-T271), IFN-gamma (4S.B3), IL-2 (MQ1-17H12), TNF-alpha (MAb11), IL-17A (BL168), CD69 (FN50), PD-1 (EH12.2H7), and TIM-3 (F38-2E2) (Biolegend) were used to evaluate T cell phenotype. Recombinant human GPC3 protein was conjugated to AlexaFluor 647 using NHS ester chemistry and used to detect CAR+ T cells. For all flow cytometry experiments, dead cells were excluded with Fixable Viability Dye eFluor 780 (Fisher Scientific, Cat# 50-169-66). For ex vivo phenotypic studies, rat anti-mouse antibodies to CD45 (30-F11) and CD11b (M1/70) (Biolegend) were used to exclude murine cells from analysis.
To evaluate intracellular cytokine production, CAR T cells were incubated with GPC3+ HepG2 target cells at a 1:1 effector-to-target cell ratio in the presence of brefeldin A (5 μg/mL) and monensin (2 μM) protein transport inhibitors. The co-cultures were incubated for 6 hours at 37°C and 5% CO2 prior to cell staining using the Cytofix/Cytoperm Fixation Kit (BD Biosciences, Cat# 554715/555028).

Hickman T.L., Choi E., Whiteman K.R., Muralidharan S., Pai T., Johnson T., Parikh A., Friedman T., Gilbert M., Shen B., Barron L., McGinness K.E., Ettenberg S.A., Motz G.T., Weiss G.J, & Jensen-Smith A. (2022). BOXR1030, an anti-GPC3 CAR with exogenous GOT2 expression, shows enhanced T cell metabolism and improved anti-cell line derived tumor xenograft activity. PLoS ONE, 17(5), e0266980.

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Intracellular Cytokine Profiling of NK Cells

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NK cells were subjected to intracellular cytokine-staining assay following 1 h incubation with monensin (2 mM, BD) or to evaluate the production of IFNγ, after an overnight stimulation with PMA (10 ng/ml, Sigma-Aldrich, Milan, Italy) and Ionomycin (500 ng/ml, Sigma-Aldrich) plus monensin (2 mM, BD). Briefly, following staining with anti-human mAbs CD3-PerCP, CD56-APC, and CD16-FITC (Miltenyi Biotec), cells were permeabilized and fixed using the Cytofix/Cytoperm fixation kit (BD), according to the manufacturer's instructions and finally stained with different anticytokine PE-conjugated mAbs (VEGF, SDF-1, perforin, osteopontin, IL-8, or IFNγ, all from Miltenyi Biotec).

Bosi A., Zanellato S., Bassani B., Albini A., Musco A., Cattoni M., Desio M., Nardecchia E., D'Urso D.G., Imperatori A., Dominioni L., Noonan D.M., Mortara L, & Bruno A. (2018). Natural Killer Cells from Malignant Pleural Effusion Are Endowed with a Decidual-Like Proangiogenic Polarization. Journal of Immunology Research, 2018, 2438598.

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