Foxp3 fixation permeabilization buffer set

Manufactured by Thermo Fisher Scientific

The Foxp3 fixation/permeabilization buffer set is a laboratory reagent used to prepare cells for intracellular staining and flow cytometric analysis of Foxp3 and other intracellular markers. The set includes a fixation/permeabilization concentrate and a permeabilization buffer.

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Lab products found in correlation

Efluor 506, by Thermo Fisher Scientific (1 mentions) Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Il 17a, by Miltenyi Biotec (1 mentions) Cd45ro clone uchl1, by Thermo Fisher Scientific (1 mentions) Macsquant analyzer 10, by Miltenyi Biotec (1 mentions) Facsdiva v8, by BD (1 mentions) Live dead fixable near ir dye, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Permeabilization buffer (703 citations) Fixation/permeabilization buffer (268 citations) Fixation buffer (92 citations) Foxp3 Fixation/Permeabilization buffer (51 citations) FoxP3 permeabilization buffer (21 citations) FoxP3 fixation buffer (19 citations) FoxP3 buffer set (19 citations) FOXP3 Fixation/Permeabilization set (2 citations)

4 protocols using foxp3 fixation permeabilization buffer set

Phenotypic Analysis of PBMCs

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PBMCs from patients and HDs were phenotypically analyzed by 14-color flow-cytometry. Cryopreserved cells were thawed and resuspended in phosphate buffer saline (PBS), supplemented with 0,5% bovine serum albumin and 2mM ethylenediaminetetraacetic acid. Two million cells were stained for surface markers described in Supplementary Table 1. After washing with PBS, cells were stained with a fixable viability dye (eFluor 506; eBioscience). Finally, PBMCs were washed, fixed and permeabilized using the Foxp3 fixation/permeabilization buffer set (eBioscience) to allow intracellular staining of T-bet. Data were acquired on a BD LSR-II flow-cytometer and analyzed using FlowJo software 10.7 (Tree Star, Ashland, Oregon). Gates were based on fluorescence-minus-one (FMO) controls.

Wilbrink R., Spoorenberg A., Arends S., van der Geest K.S., Brouwer E., Bootsma H., Kroese F.G, & Verstappen G.M. (2021). CD27-CD38lowCD21low B-Cells Are Increased in Axial Spondyloarthritis. Frontiers in Immunology, 12, 686273.

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Regulatory T Cell Depletion Protocol

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Information of all antibodies are shown in Supplementary Table 1. Fixable Viability Dye eFluor 506 (eBioscience) was used to exclude dead cells. FoxP3 Fixation/Permeabilization buffer set was purchased from eBioscience. DT was purchased from Sigma-Aldrich. Treg depletion was obtained as described in (ref. 37 (link)). Briefly, in transplantation experiments daily 50 mg kg^–1 DT was injected intraperitoneally at days −2 and −1 before transplantation; for Treg depletion in untreated mice 50 mg kg^–1 DT was injected intraperitoneally every other day for five times. In both the cases efficiency of Treg depletion was checked the day after the last DT injection by PB FACS analysis.

Pierini A., Nishikii H., Baker J., Kimura T., Kwon H.S., Pan Y., Chen Y., Alvarez M., Strober W., Velardi A., Shizuru J.A., Wu J.Y., Chiba S, & Negrin R.S. (2017). Foxp3+ regulatory T cells maintain the bone marrow microenvironment for B cell lymphopoiesis. Nature Communications, 8, 15068.

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Multiparameter T-cell Phenotyping

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The following fluorescence conjugated antibodies, from Miltenyi Biotec were used: CD4 (clone: M-T466), CD45RA (clone: REA562), CD196 (clone: REA190), CD127 (clone: REA614), IL-17A (clone: CZ8–23G1). Conjugated antibodies against the following were from eBiosciences: CD25 (clone BC96), CTLA4 (clone 14D3), CD161 (clone HP-3G10), FOXP3 (clone 236A/E7), IL-10 (clone JES3-9D7) and CD45RO (clone UCHL1). Conjugated antibodies against the following were from R&D systems: RORγT (clone AFKJS-9) and IL-1R1 (Catalog number FAB269F). Antibodies against TGF-β (clone TW4–9E7) and CD126 (clone M5) was from BD Biosciences. Intracellular antigens were detected using Foxp3 Fixation/Permeabilization buffer set (eBiosciences) according to the manufactures instruction after blocking with anti-human FcR (Miltenyi Biotec). Cells were acquired immediately after staining on MACSQuant 8-color flow cytometer and data were analyzed using MACS quant analyzer 10 (Miltenyi Biotec). Unstained cells and Isotype-matched antibodies (directly conjugated) were used as controls. Dead cells were excluded by using Live/Dead fixable aqua dead cell stain kit (Thermo Fisher Scientific).

Rajendran M., Looney S., Singh N., Elashiry M., Meghil M.M., El-Awady A.R., Tawfik O., Susin C., Arce R.M, & Cutler C.W. (2019). Systemic Antibiotic Therapy Reduces Circulating Inflammatory Dendritic Cells and Treg-Th17 Plasticity in Periodontitis.. Journal of immunology (Baltimore, Md. : 1950), 202(9), 2690-2699.

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Flow Cytometry of Cryopreserved AML Cells

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For MFC, cryopreserved or fresh mononuclear cells of patients with CD117⁺CD34⁺ AML and PB-derived mononuclear cells from HDs were used. Cells were stained with the LIVE/DEAD Fixable Near-IR dye (Thermo Fisher) for exclusion of dead cells and incubated with appropriate fluorochrome-conjugated antibodies (online supplemental table 1). For intracellular staining, the cells were permeabilized using the FOXP3 Fixation/Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions. Compensation controls were measured using single-stained Comp Beads (BD Biosciences). All samples were run on a BD FACSymphony V.A3 with FACS Diva V.8 (BD Biosciences). Statistical analyses are described in the online supplemental methods 3.

Brauneck F., Fischer B., Witt M., Muschhammer J., Oelrich J., da Costa Avelar P.H., Tsoka S., Bullinger L., Seubert E., Smit D.J., Bokemeyer C., Ackermann C., Wellbrock J., Haag F, & Fiedler W. (2022). TIGIT blockade repolarizes AML-associated TIGIT+ M2 macrophages to an M1 phenotype and increases CD47-mediated phagocytosis. Journal for Immunotherapy of Cancer, 10(12), e004794.

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