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Fixation and permeabilization buffer kit

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The Fixation and Permeabilization Buffer Kit is a laboratory product designed to prepare samples for flow cytometry analysis. The kit contains a fixation buffer and a permeabilization buffer, which are used to fix and permeabilize cells, respectively, in order to enable the detection of intracellular targets.

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Lab products found in correlation

Cytofix cytoperm kit, by BD (3 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions) Brefeldin a, by BD (2 mentions) Ionomycin, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by Thermo Fisher Scientific (1 mentions) Anti cd16 32 clone 2.4g2, by BioXCell (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Lsrfortessa, by BD (1 mentions) Il 17a, by Thermo Fisher Scientific (1 mentions) Ionomycin, by Merck Group (1 mentions) Facscanto 2, by BD (1 mentions) Golgistop, by BD (1 mentions) Live dead fixable yellow dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Cd45 2d1, by Thermo Fisher Scientific (1 mentions) Pd 1 mih4, by Thermo Fisher Scientific (1 mentions) Cd62l, by Thermo Fisher Scientific (1 mentions) Mouse inflammation kit, by BD (1 mentions) Pd l1, by Thermo Fisher Scientific (1 mentions) Lag 3 17b4, by Enzo Life Sciences (1 mentions) Cd3 sk7, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Permeabilization buffer (703 citations) Fixation/permeabilization buffer (268 citations) Fixation/permeabilization kit (189 citations) Fixation buffer (92 citations) Fixation and permeabilization kit (29 citations) Fixation/permeabilization buffer kit (22 citations)

4 protocols using fixation and permeabilization buffer kit

1

Multiparametric Flow Cytometry Analysis

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Flow cytometry was performed using an LSRFortessa (BD) and analyzed on FlowJo v10 (BD). Fc receptors were blocked using anti-CD16/32 (clone 2.4G2, Bio X Cell). Antibodies specific for mouse and human antigens (Supp. Table S1) conjugated to various fluorochromes were used to stain single cells. If necessary, cells were fixed using the eBioscience Fixation and Permeabilization Buffer Kit (cat# 88–8824-00) as per the manufacturer’s protocol. Intracellular cytokine staining was performed with the BD Cytofix/Cytoperm Kit (cat# 554714), as per the manufacturer’s protocol. For intracellular IFNγ and TNF staining, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich, cat# 79346) and 1 μM ionomycin (Life Technologies, cat# I24222) and 1 μg/mL LPS (Sigma-Aldrich, cat# L6529) and incubated at 37oC, 5% CO2 in the presence of 10 μg/mL brefeldin A (Affymetrix, Cat# 00–4506-51) for 4 hours.
Liu M., Etherington M.S., Hanna A., Medina B.D., Vitiello G.A., Bowler T.G., Param N.J., Levin L., Rossi F, & DeMatteo R.P. (2021). Oncogenic KIT modulates type I interferon–mediated antitumor immunity in GIST. Cancer immunology research, 9(5), 542-553.
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2

Comprehensive Immune Cell Profiling

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Cell suspensions obtained from mouse organs were preincubated with FcγR-specific blocking antibodies (eBioscience, San Diego, Calif) and stained with the following antibodies from eBioscience: TCR-γδ (UC7-13D5), CD45 (30-F11), CD4 (RM4-5), CD8a (53-6.7), CCR9 (CW-1.2), CD103 (2E7), CD11C (N418), MHCII (M5/114.15.2), CCR4 (2G12), IL-17A (17B7) and IFN-γ (XMG1.2). Dead cells exclusion was performed with Aqua Cell Stain kit (Invitrogen, Thermo Fisher Scientific). For intracellular staining, cells were stimulated for 4 hours with phorbol 12-myristate 13-acetate (5 ng/ml; Sigma-Aldrich) and ionomycin (1 μg/ml; Sigma-Aldrich). Golgi stop (1000×; BD, Franklin Lakes, NJ) was added during the last 3 hours of stimulation. Cell fixation and permeabilization was performed using the Fixation and Permeabilization Buffer kit (eBioscience). Human cells were stained with the following antibodies from BioLegend (San Diego, Calif): CD4 (OKT4), CD8 (RPA-T8), CD45RA (HI100), CD45RO (UCHL1), CLA (HECA-452), CCR4 (L291H4). Fluorescence-activated cell sorting (FACS) data were acquired with FACSCanto II (BD) and analyzed with FlowJo software (version 9.9.6; Tree Star, BD).
Rigoni R., Fontana E., Dobbs K., Marrella V., Taverniti V., Maina V., Facoetti A., D’Amico G., Al-Herz W., Cruz-Munoz M.E., Schuetz C., Gennery A.R., Garabedian E.K., Giliani S., Draper D., Dbaibo G., Geha R.S., Meyts I., Tousseyn T., Neven B., Moshous D., Fischer A., Schulz A., Finocchi A., Kuhns D.B., Fink D.L., Lionakis M.S., Swamydas M., Guglielmetti S., Alejo J., Myles I.A., Pittaluga S., Notarangelo L.D., Villa A, & Cassani B. (2020). Cutaneous barrier leakage and gut inflammation drive skin disease in Omenn syndrome. The Journal of Allergy and Clinical Immunology, 146(5), 1165-1179.e11.
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3

Multiparameter Flow Cytometry Analysis

Cited 1 time
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Human-specific antibodies were purchased from BD Biosciences (CD3: SP34-2; CD4: RPA-T4) or BioLegend (CD8: HIT8a; PD1: EH12.2H7; TIM-3: F38-2E2; LAG3-: 11C3C65; CTLA-4: BNI3; TIGIT: A15153G; CD39: A1; IL-2: MQ1-17H12; TNF-α: MAb11; IFN-γ: 4S.B3 (San Diego, CA). Mouse-specific antibodies were purchased from BD Biosciences (CD3: 145-2C11; CD4: RM4-5; CD8: 53-6.7; PD1: J43; Lag-3: C9B7W; TIGIT: 1G9; CD244.2: 2B4; CD160: CNX46-3; CTLA4: UC10-4F10) or Biolegend (IFN-γ: XMG1.2; IL-2: JES6-5H4; TNF-α: MP6-XT22; Tim-3: RMT3-23; CD39: Duha59; BTLA: 8F4). Appropriate isotype controls were used when applicable. LIVE/DEAD ® Fixable Yellow Dead Cell Stain Kit (ThermoFischer) was used to exclude dead cells. Intracellular staining was performed using the eBioscience Fixation and Permeabilization Buffer kit. For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 50nM) and ionomycin (500 nM) for 4–6h at 37°C, 5% CO2 in the presence of 1 ug/ml brefeldin A (BD biosciences). Surface staining was performed and cells were fixed and permeabilized with the BD Cytofix/Cytoperm kit and stained for IFN-γ, TNF-α, and IL-2. Tetramers for mODC1 were generated and used as described previously (21 (link)).
Woroniecka K., Chongsathidkiet P., Rhodin K., Kemeny H., Dechant C., Farber S.H., Elsamadicy A.A., Cui X., Koyama S., Jackson C., Hansen L.J., Johanns T.M., Sanchez-Perez L., Chandramohan V., Yu Y.R., Bigner D.D., Giles A., Healy P., Dranoff G., Weinhold K.J., Dunn G.P, & Fecci P.E. (2018). T Cell Exhaustion Signatures Vary with Tumor Type and are Severe in Glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(17), 4175-4186.
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4

Multi-Marker Flow Cytometry Analysis

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Human-specific antibodies were purchased from BD Biosciences (CD45, 2D1; CD3, SK7; CD4, RPA-T4; PD-1, MIH4), eBioscience (CD8, RPA-T8), BioLegend (PD-L1, 29E.2A3), R&D Systems (TIM-3, 344823), and Enzo Life Sciences (LAG-3, 17B4). Mouse-specific antibodies were purchased from BD Biosciences (CD3, 125-2C11; CD4, GK1.5; CD44, IM7; CD62L, MEL-14; PD-L1, MIH5; CD69, H1.2F3; IFN-γ, XMG1.2; TNF, MP6-XT22), eBioscience (CD8, 53-6.7; PD-1, J43; PD-L2, 122; Foxp3, FJK-16s; Ki67, SolA15; Granzyme B, NGZB), and BioLegend (CD45, 30-F11; PD-L1, 10F.9G2; CD25, PC61). Appropriate isotype controls were used where applicable. A viability dye was typically used to exclude dead cells. Intracellular staining was performed using the eBioscience Fixation and Permeabilization Buffer Kit. For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) and ionomycin (750 ng/ml) for 4 h at 37°C, 5% CO2 in the presence of 1 μg/ml brefeldin A (BD Biosciences). Surface staining was performed and cells were fixed and permeabilized with the BD Cytofix/Cytoperm Kit and stained for IFN-γ and TNF- α. Supernatant cytokines were measured by cytometric bead array according to the manufacturer’s instructions (Mouse Inflammation Kit; BD Biosciences). Data were acquired using a BD FACSAria or Fortessa LSR flow cytometer and analyzed using FlowJo software (Tree Star).
Seifert A.M., Zeng S., Zhang J.Q., Kim T.S., Cohen N.A., Beckman M.J., Medina B.D., Maltbaek J.H., Loo J.K., Crawley M.H., Rossi F., Besmer P., Antonescu C.R, & DeMatteo R.P. (2016). PD-1/PD-L1 blockade enhances T cell activity and antitumor efficacy of imatinib in gastrointestinal stromal tumors. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(2), 454-465.
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