Phosflow perm wash buffer 1

Manufactured by BD

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Phosflow™ Perm/Wash Buffer I is a laboratory reagent designed for cell permeabilization and washing. It is used to facilitate the intracellular staining of phosphorylated proteins for flow cytometry analysis.

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Lab products found in correlation

Cytofix fixation buffer, by BD (3 mentions) Facscalibur, by BD (2 mentions) Lsr fortessa instrument, by BD (1 mentions) Cytofix cytoperm, by BD (1 mentions) Lsrii flow cytometer, by BD (1 mentions) A11008, by Thermo Fisher Scientific (1 mentions) N5413, by Merck Group (1 mentions) Accuri c6 flow cytometer, by BD (1 mentions) Dispase, by Thermo Fisher Scientific (1 mentions) Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions) Alexa 488, by Cell Signaling Technology (1 mentions) Anti afp, by R&D Systems (1 mentions) Anti β catenin, by GeneTex (1 mentions) Phosflow protocol for human pbmcs, by BD (1 mentions) Phosflow lyse fix buffer, by BD (1 mentions) Phosflow protocols for mouse splenocytes, by BD (1 mentions)

Spelling variants of this product

Perm/Wash buffer (1124 citations) Perm/Wash (346 citations) BD Phosflow (39 citations) Perm buffer (32 citations) Wash buffer (27 citations) Perm/Wash Buffer I (23 citations) BD Phosflow Perm Buffer (15 citations) Perm Buffer I (3 citations) BD Phosflow Perm/wash I (2 citations) BD Perm/Wash I (2 citations)

7 protocols using phosflow perm wash buffer 1

Flow Cytometric Profiling of Immune Cells

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Flow cytometric analysis was performed in accordance to published guidelines (30 (link)). Single cell suspensions of bone marrow (BM), mesenteric lymph nodes (mes-LN) and mediastinal lymph nodes (med-LN) were generated by mechanical disruption. Erythrocytes in the BM were lysed with ACK-buffer (0.15M NH₄Cl, 1mM KHO₃, 0.1mM Na₂EDTA) and all cells resuspended in FACS buffer (PBS, 2% FCS, 1 mg/mL NaN₃). For staining of IgE-expressing cells cytophilic IgE was efficiently removed by short treatment with acetate buffer as described (26 (link)). Fc receptors were blocked with anti-mouse CD16/CD32 mAb (BioXcell) for 5 min at RT and the primary antibodies were incubated for 25 min at 4°C in the dark (for list of antibodies used refer to Supplementary Table S1).
As secondary staining streptavidin conjugated to BUV 395 was added and the cells were incubated for 20 min at 4°C in the dark. For PCs and GC B cells during primary and secondary infection IgA, IgE and IgG1 were instead intracellularly stained using Cytofix/Cytoperm™ (BD Bioscience) and Phosflow™ Perm/Wash Buffer I (BD Bioscience). Cells were analyzed on a BD LSR-Fortessa instrument (BD Bioscience).

Haase P., Schäfer S., Gerlach R.G., Winkler T.H, & Voehringer D. (2022). B cell fate mapping reveals their contribution to the memory immune response against helminths. Frontiers in Immunology, 13, 1016142.

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Single-Cell Characterization of Neural Stem Cells

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Single cell suspensions were collected from the scaffolds. A fixable yellow dead stain kit (Invitrogen) was used to identify dead cells. The cells were fixed with 4% paraformaldehyde for 10 min on ice, permeabilized in 1% v/v Triton-X 100, washed with PhosFlow Perm/Wash Buffer I (BD Biosciences, San Jose, CA, USA), and resuspended in a buffer. Antibodies PE anti-nestin (BDB561230), PerCP-Cy5.5 anti-SOX1 (BDB561549), Alexa Fluor 647 anti-SOX2 (BDB560302), and Alexa Flour 488 anti-PAX6 (BD561664) were added, incubated on ice for 1 h, and washed with a buffer. The cells were resuspended in 200 μL of buffer and stained with DAPI. All samples were analyzed on a LSRII flow cytometer (BD Biosciences), and the data were plotted using FlowJo software Version 9.9.4 (Tree Star Inc., Ashland, OR, USA).

Ando Y., Chang F.C., James M., Zhou Y, & Zhang M. (2023). Chitosan Scaffolds as Microcarriers for Dynamic Culture of Human Neural Stem Cells. Pharmaceutics, 15(7), 1957.

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Nestin Expression Analysis in Differentiated Cells

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Following 9 days of differentiation in vitro, the cultures were trypsinized into single cells and washed once with FACS buffer [PBS plus 1% fetal bovine serum (FBS)]. The cells were fixed in Cytofix™ Fixation Buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature. Following 3 washes with FACS buffer, permeabilization was performed using Phosflow™ Perm/Wash Buffer I (BD Biosciences) for 10 min at 4°C. The cells were then incubated with primary antibody against Nestin (1:200; N5413; Sigma-Aldrich, St. Louis, MO, USA) diluted in Phosflow™ Perm/Wash Buffer I for 15 min at 37°C. The cells were washed with FACS buffer 3 times, followed by incubation with secondary antibody (1:500; A11008; Life Technologies, Eugene, OR, USA) in FACS buffer for 15 min at 37°C. The cells were washed and resuspended in PBS. Analyses were performed using an Accuri C6 flow cytometer (BD Biosciences) and FlowJo software.

Zhang N., Lyu Y., Pan X., Xu L., Xuan A., He X., Huang W, & Long D. (2017). miR-146b-5p promotes the neural conversion of pluripotent stem cells by targeting Smad4. International Journal of Molecular Medicine, 40(3), 814-824.

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Multimarker Profiling of Hepatic Cells

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Cultured cells were dissociated using dispase (Thermo Fisher Scientific) and a cell dissociation buffer (Thermo Fisher Scientific). Cells fixed with Cytofix Fixation Buffer (BD, USA) were washed with PhosFlow Perm/Wash Buffer I (BD) and incubated with primary antibodies (anti-HNF4α [GeneTex, USA], anti-CK19 [GeneTex], anti-AFP [R&D Systems, USA], anti-albumin [R&D Systems], anti-β-catenin [GeneTex], anti-Numb [R&D Systems], anti-Notch 1 ICD [R&D Systems], and anti-Hes5 [Bioss, USA] antibodies at a dilution of 1:400) at 4 °C overnight. After the cells were washed, they were incubated with Alexa 488 (Cell Signaling Technology, USA)- and phycoerythrin (Southern Biotech, USA)-conjugated anti-rabbit and anti-mouse IgG, respectively at 4 °C overnight. Then, the cells were washed again and passed through a 45-μm filter, and were subjected to flow cytometric analysis using a FACS Calibur (BD) flow cytometer according to the manufacturer's instructions. For flow cytometric analysis, the proportion of immunopositive cells was determined using the antibodies mentioned above. The data, which were normalized to the population of HNF4α⁺ cells, were represented as the relative value compared to that obtained following solvent control treatment.

Morita A., Omoya Y., Ito R., Ishibashi Y., Hiramoto K., Ohnishi S., Yoshikawa N, & Kawanishi S. (2021). Glycyrrhizin and its derivatives promote hepatic differentiation via sweet receptor, Wnt, and Notch signaling. Biochemistry and Biophysics Reports, 28, 101181.

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Phosphorylation of ERK1/2 in PBMCs

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The phosphorylation of ERK1/2 was analyzed using the BD PhosFlow Protocol for Human PBMCs. After isolation, PBMCs were incubated for 30 min in complete medium (Table S5) without or with DARA at 1 µg/ml. Afterwards, the samples were washed and resuspended in complete medium and left to equilibrate at 37°C for 20 min. An equal volume of prewarmed complete medium containing either α-Ig, CpG-B, or IL-2 (as described in Materials and methods) were added and the samples were incubated for 10, 30, or 60 min at 37°C, or left unstimulated (control). The samples were then fixed with BD Cytofix Fixation Buffer (BD Biosciences) for 10 min at 37°C, washed twice in Phosflow Perm/Wash Buffer I (BD Biosciences), and stained for pERK1/2 (Table S4) for 60 min at room temperature. Surface and intracellular stainings with other mAbs were performed as required by the experimental design. Samples were washed and resuspended in Phosflow Perm/Wash Buffer I for flow cytometric analysis.

Camponeschi A., Kläsener K., Sundell T., Lundqvist C., Manna P.T., Ayoubzadeh N., Sundqvist M., Thorarinsdottir K., Gatto M., Visentini M., Önnheim K., Aranburu A., Forsman H., Ekwall O., Fogelstrand L., Gjertsson I., Reth M, & Mårtensson I.L. (2022). Human CD38 regulates B cell antigen receptor dynamic organization in normal and malignant B cells. The Journal of Experimental Medicine, 219(9), e20220201.

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BAX Protein Expression Analysis

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Approximately 1-2×10⁶ cells were pelleted by centrifugation, resuspended in Lyse Fix Buffer I (BD BioSciences) for 10 minutes, washed once with PBS-D, then permeabilized with 1x BD Phosflow Perm Wash Buffer I. BAX antibody or isotype control was added to 1×10⁵ fixed/permeabilized cells. Samples were incubated for 30 min at room temperature in the dark and washed, followed by the addition of an APC-conjugated F(ab’)2. After incubation for 30 min at room temperature, the cells were washed twice and the samples stored in Fix buffer I. Samples were then analyzed using a BD FACSCalibur and CellQuest Pro software [18 ].

Tahir S.K., Smith M.L., Hessler P., Rapp L.R., Idler K.B., Park C.H., Leverson J.D, & Lam L.T. (2017). Potential mechanisms of resistance to venetoclax and strategies to circumvent it. BMC Cancer, 17, 399.

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Phosphoflow Protocol for Lung Cells

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Lung tissue was processed as described above, except that the digestion with 2 mg mL -1 of collagenase IV was reduced to 30 min at 37 °C in a shaker. Phosphoflow staining was performed according to Protocol I described in BD Phosflow Protocols for Mouse Splenocytes. Briefly, following viability dye staining, samples were fixed with BD Phosflow Lyse/Fix Buffer for 11 min at 37 °C, permeabilized with BD Phosflow Perm/Wash buffer I for 30 min at RT, then stained with antibody mixture for 1 h at RT.

Chu K.L., Batista N.V., Wang K.C., Zhou A.C, & Watts T.H. (2019). GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T-cell accumulation and tissue-resident memory T-cell formation. Mucosal immunology, 12(2).

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