Phosflow protocol 3

Manufactured by BD

BD Phosflow Protocol III is a laboratory equipment product designed for the analysis of cellular signaling pathways. The core function of this product is to enable the detection and measurement of phosphorylated proteins within cells.

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Lab products found in correlation

Perm buffer 3, by BD (2 mentions) Pharmingen stain buffer, by BD (1 mentions) Phosflow perm buffer 3, by BD (1 mentions) Lyse fix buffer, by BD (1 mentions) Cytofix buffer, by BD (1 mentions) Facsaria 2, by BD (1 mentions) Glut1, by Abcam (1 mentions) Phospho s6, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

BD Phosflow (39 citations) BD Phosflow Protocol (7 citations)

4 protocols using phosflow protocol 3

Monocyte NF-κB p65 Phosphorylation Assay

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CD14⁺ monocytes from healthy donors were magnetically isolated as described above and pretreated with 10 and 100 μM of laquinimod and activated with LPS (1 μg/mL) for 30 minutes in vitro. Monocyte fixation and permeabilization were performed according to BD Phosflow Protocol III for human peripheral blood mononuclear cells (BD Phosflow Protocol III 2011). To ensure permeabilization of both cell and nuclear membranes, the BD Phosflow Perm Buffer III was used. After permeabilization, the cell samples were washed twice with BD Pharmingen Stain Buffer and resuspended in 100 μL of the buffer. Then, 5 μL aliquots of anti-NF-κB p65-PE-CF594 were added to LPS-treated samples and respective reference samples without stimulus. After incubation for 30 minutes in the dark, samples were analyzed by flow cytometry to assess phosphorylation of p65.

Engel S., Jolivel V., Kraus S.H., Zayoud M., Rosenfeld K., Tumani H., Furlan R., Kurschus F.C., Waisman A, & Luessi F. (2020). Laquinimod dampens IL-1β signaling and Th17-polarizing capacity of monocytes in patients with MS. Neurology® Neuroimmunology & Neuroinflammation, 8(1), e908.

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Phospho-STAT1 Staining in Th9 Cells

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For staining of phosphorylated STAT1, human Th9 cells were harvested after 48 h of culture, rested for 4 h and treated with rhIFN-γ (100 ng ml⁻¹) for 20 min. Then the cells were fixed and permeabilized according to BD Phosflow Protocol III using Lyse/Fix Buffer (557870, BD) and Perm Buffer III (558050, BD) and stained with anti-human phospho-STAT1 (Y701)-eFluor660 (KIKSI0803, eBioscience, 1:100).

Campos Carrascosa L., Klein M., Kitagawa Y., Lückel C., Marini F., König A., Guralnik A., Raifer H., Hagner-Benes S., Rädler D., Böck A., Kang C., Lohoff M., Garn H., Schaub B., Berberich-Siebelt F., Sakaguchi S., Bopp T, & Huber M. (2017). Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4. Nature Communications, 8, 15366.

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Intracellular AKT Phosphorylation Assay

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The intracellular protein staining was performed according to BD phosflow Protocol III. Briefly, after CXCL12 stimulation, the cells were fixed by adding pre-warmed BD Cytofix buffer. For permeabilization, the fixed cells were permeabilized by incubation of chilled BD Perm buffer III for 30 minutes. After permeabilization, the cells were incubated with PE- Mouse anti-AKT (catalogue number 554655) for 1 hour.

Pak H.K., Gil M., Lee Y., Lee H., Lee A.N., Roh J, & Park C.S. (2015). Regulator of G Protein Signaling 1 Suppresses CXCL12-Mediated Migration and AKT Activation in RPMI 8226 Human Plasmacytoma Cells and Plasmablasts. PLoS ONE, 10(4), e0124793.

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Regulatory T Cell Isolation and Phenotyping

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CD4⁺ T cells from spleens and lymph nodes (inguinal, axiliary and cervical) were enriched using the CD4⁺ T cell negative selection kit (ebioscience) and sorted to over 95% purity on a FACSAria II (BD) for YFP+ CD4+ Tregs and YFP- CD4+ CD62L^hi CD44^lo CD25 –ve naïve T cells. PhosphoFlow, intracellular staining for PTEN and Glut1 was performed using the BD Phosflow™ protocol III with antibodies for PTEN (BD, Clone A2B1), phospho Akt 473 (Cell signaling), phospho S6 (Cell Signaling) and Glut1 (abcam). The affinity pure (F-(ab’)2) fragment donkey anti-rabbit IgG (Jackson Immunoresearch) was used as secondary antibody for staining pAkt473 and pS6. The following antibodies to ICOS (Clone 7E.17G9) and CTLA-4 (Clone UC10–4B9) were also used.

Priyadharshini B., Loschi M., Newton R.H., Zhang J.W., Finn K.K., Gerriets V.A., Huynh A., Rathmell J.C., Blazar B.R, & Turka L.A. (2018). TGF-β and PI3K Signals Modulate Distinct Metabolism of Treg Subsets. Journal of immunology (Baltimore, Md. : 1950), 201(8), 2215-2219.

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