Phosflow antibody

Manufactured by BD

BD Phosflow antibodies are flow cytometry reagents designed to detect the phosphorylation state of specific cellular proteins. They provide a direct measurement of cellular signaling pathway activation.

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Lab products found in correlation

Live dead aqua, by Thermo Fisher Scientific (1 mentions) Cd19 apc, by BD (1 mentions) Cd5 fitc, by BioLegend (1 mentions) Il 10, by R&D Systems (1 mentions) Cytofix buffer, by BD (1 mentions) Phosflow fixation buffer, by BD (1 mentions) Anti phospho p38 pe, by BD (1 mentions) Phosflow perm buffer, by BD (1 mentions) Facscalibur, by BD (1 mentions) Phosflow perm buffer 3, by BD (1 mentions) Phosflow fix buffer 1, by BD (1 mentions) Pharmingen stain buffer, by BD (1 mentions) Phosflow, by BD (1 mentions)

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BD Phosflow (39 citations)

6 protocols using phosflow antibody

Analyzing STAT3 Phosphorylation in CLL and T Cells

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To analyze S727-STAT3 phosphorylation in CLL cells, we cultured PBMCs from CLL patients in the presence or absence of the STAT3 inhibitor cucurbitacin (0.05 µM) for 2 h or lenalidomide (10 µM) for 2 or 24 h at 37^oC. Cells were stained for 30 min with Live/Dead-Aqua (Invitrogen). Samples were stimulated with 250 ng/ml of CXCL12 for 20 min at 37^oC, and then fixed for 10 min in the dark. After one wash and 20 min of surface staining with CD19-APC (BD Biosciences) and CD5-FITC (BioLegend) antibodies, the cells were washed, permeabilized (Phospho-Epitopes Exposure kit-Beckman Coulter kit), and stained with p-S727-STAT3-PE mAb Phosflow antibody (BD Biosciences) for 30 min at room temperature.
To analyze Y705-STAT3 phosphorylation in T cells, we incubated healthy donor PBMC for 20 min at 37^°C with IL-10 (10 ng/ml) (R&D) or with supernatants collected from CXCL12-treated CLL cells (supernatant was harvested after CLL cells were treated with 250 ng/ml of CXCL12 for 48 h). Cells were fixed and stained with anti-CD3-v450 (BD Biosciences) and phosphorylated Y705-STAT3-PE mAb Phosflow antibody (BD Biosciences), following the protocol described above. To examine the effect of lenalidomide on Y705-STAT3 phosphorylation in T cells we pre-incubated the T cells for 2 h with lenalidomide (10 µM) before adding IL-10/CLL cells supernatant.

Shaim H., Estrov Z., Harris D., Hernandez Sanabria M., Liu Z., Ruvolo P., Thompson P.A., Ferrajoli A., Daher M., Burger J., Muftuoglu M., Imahashi N., Li L., Liu E., Alsuliman A.S., Basar R., Nassif Kerbauy L., Sobieski C., Gokdemir E., Kondo K., Wierda W., Keating M., Shpall E.J, & Rezvani K. (2018). The CXCR4–STAT3–IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia and Is Modulated by Lenalidomide. Frontiers in Immunology, 8, 1773.

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Intracellular Signaling Pathway Analysis

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Treated PBMCs were washed and pDCs were stained as described. CpG-mediated induction of pIRF7, pTBK1, p65 (NFκB), pIKKγ, pS473-AKT, and pT308-AKT were determined using Phosflow™ antibodies and the harsh detergent method by BD Biosciences©. In brief, cells were fixed using BD cytofix buffer for 10 min at 37 °C then permeabilized using 1 × of perm buffer IV™, stained for 1 h under continuous motion using FACS buffer and 5% Human AB serum, washed 3× with 0.5× perm buffer, and analyzed by flow cytometry.

Henriquez J.E., Crawford R.B, & Kaminski N.E. (2019). Suppression of CpG-ODN-mediated IFNα and TNFα response in human plasmacytoid dendritic cells (pDC) by cannabinoid receptor 2 (CB2)-specific agonists. Toxicology and applied pharmacology, 369, 82-89.

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Phosphorylation of IRF7 in pDCs

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Treated PBMCs were washed and pDCs were stained as described. pIRF7 levels were determined using Phosflow™ antibodies and the harsh detergent method by BD Biosciences©. In brief, cells were fixed using BD cytofix buffer for 10 min at 37°C then permeabilized using 1x of perm buffer IV™, stained for 1 hr under continuous motion using FACS buffer and 5% Human AB serum, washed 3X with 0.5x perm buffer, and analyzed by flow cytometry.

Henriquez J.E., Rizzo M.D., Schultz M.A., Crawford R.B., Gulick P, & Kaminski N.E. (2017). Δ9-Tetrahydrocannabinold (THC) suppresses secretion of IFNα by plasmacytoid dendritic cells (pDC) from healthy and HIV-infected individuals. Journal of acquired immune deficiency syndromes (1999), 75(5), 588-596.

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ARPE-19 Cellular Signaling Responses

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Confluent ARPE-19 cells were cultured with serum-free DMEM/F12 at 37°C in 5% CO₂ for 24 h. Then the cells were incubated in medium containing additional 20 mM NaCl or 40 mM NaCl in the presence of 100 ng/mL LPS for 20 min. Cells were harvested and stained for intracellular phosphorylated ERK-1/2, P38, Akt, JNK, and NF-κB using BD Phosflow antibodies according to the protocol of the manufacturer (BD Biosciences). Before detection, ARPE-19 cells were fixed in BD Phosflow Fixation Buffer (BD Biosciences) for 10 min at 37°C and permeabilized in BD Phosflow Perm Buffer (BD Biosciences) for 30 min on ice. Anti-phospho-Akt-Alexa Fluor 488, anti-phospho-NF-κB-Alexa Fluor 488, anti-phospho-p38-PE, anti-phospho-ERK-PE, and anti-phospho-JNK-PE (BD Biosciences) were used to stain the cells. Isotype-matched irrelevant Abs were used as controls. Phosphorylation of the five proteins for the various ARPE-19 cell treatment groups was evaluated by flow cytometry and expressed as mean fluorescence intensity (MFI).

Zhang D., Wang C., Cao S., Ye Z., Deng B., Kijlstra A, & Yang P. (2015). High-Salt Enhances the Inflammatory Response by Retina Pigment Epithelium Cells following Lipopolysaccharide Stimulation. Mediators of Inflammation, 2015, 197521.

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Intracellular Phosphorylation Profiling

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The intracellular phosphorylation level of Akt and STAT6 in dMφ after 24-h culture with JEG-3 and DSCs was analyzed using BD Phosflow antibodies, according to standard protocols.

Meng Y.H., Zhou W.J., Jin L.P., Liu L.B., Chang K.K., Mei J., Li H., Wang J., Li D.J, & Li M.Q. (2017). RANKL-mediated harmonious dialogue between fetus and mother guarantees smooth gestation by inducing decidual M2 macrophage polarization. Cell Death & Disease, 8(10), e3105-.

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Nicotine Kinase Phosphorylation in huImDCs

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To determine the effect of nicotine on kinases phosphorylation, hu-imDCs were collected by trypsination and treated with nicotine (10⁻⁷ mol/L) for 15 min. The phosphorylation of related kinase was determined by BD Phosflow. Briefly, at the end of nicotine treatment, the cells were immediately mixed with warmed BD Phosflow Fix Buffer I and incubated at 37°C for 10 min. Then, the cells were washed with BD Pharmingen Stain Buffer and permeabilized by incubation with cold BD Phosflow Perm Buffer III for 30 min on ice. After complete washes, the cells were stained with BD Phosflow antibodies and flow cytometry was done with FACSCalibur and data were analyzed with CellQuest software.

Wang Y.Y., Yang Y.W., You X., Deng X.Q., Hu C.F., Zhu C., Wang J.Y., Gu J.J., Wang Y.N., Li Q, & Gao F.G. (2015). Ex Vivo Nicotine Stimulation Augments the Efficacy of Human Peripheral Blood Mononuclear Cell-Derived Dendritic Cell Vaccination via Activating Akt-S6 Pathway. Analytical Cellular Pathology (Amsterdam), 2015, 741487.

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