Phosflow fix buffer 1

Manufactured by BD

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The BD Phosflow Fix Buffer I is a laboratory reagent used to fix cells prior to intracellular staining and flow cytometry analysis. The buffer is designed to preserve the phosphorylation state of cellular proteins, enabling the accurate detection and quantification of phosphorylated proteins within the cells.

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Fix Buffer I (63 citations) BD Phosflow (39 citations) BD Phosflow Fix Buffer (9 citations) Phosflow FIX I (2 citations)

42 protocols using phosflow fix buffer 1

Quantification of Histone Acetylation by Flow Cytometry

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This assay was performed according to the method reported by Archin et al. [25 (link)]. Briefly, cells were fixed and permeabilized with Phosflow Fix Buffer I and Phosflow Perm Buffer II (BD Biosciences) according to the manufacturer’s protocol. The cells were then washed in staining buffer (PBS with 2% FBS and 0.1% sodium azide), blocked with 8% normal goat serum (Invitrogen), and incubated with anti-AcH3 (1:200 dilution; catalog no. 06–599, Millipore) or control rabbit IgG in blocking solution for 60 minutes at room temperature. Next, the cells were washed twice with staining buffer and incubated with goat-anti-rabbit IgG FITC- or Cy3-conjugated secondary antibody (1:200 dilution; Millipore) in staining buffer for 30 minutes at room temperature in the dark. Following a final wash, the cells were analyzed by flow cytometry using a FACSAria II flow cytometer and FlowJo software (Tree Star).

Zhan X.Y., Wang N., Liu G., Qin L., Xu W., Zhao S., Qin L, & Chen X. (2014). Plasmodium infection reduces the volume of the viral reservoir in SIV-infected rhesus macaques receiving antiretroviral therapy. Retrovirology, 11, 112.

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Analyzing CRT-Mediated NK Cell Activation

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JEG-3 cells (5 × 10⁶), pretreated with oxaliplatin for 4 h at 37 °C and with 10 μg ml^–1 anti-CRT or isotype control antibody for 30 min, were incubated for 15 min at 37 °C with WT or NCR1^−/− YT cells (10⁶), pretreated for 30 min with 10 μg ml^–1 anti-NKG2D. Cells were then fixed with Phosflow Fix Buffer I and permeabilized with Phosflow Perm Buffer III (BD Bioscience) and stained for CD56, p-CD3ζ, p-Syk and p-Tyr before flow cytometry analysis on gated CD56⁺ cells.

Santara S.S., Lee D.J., Crespo Â., Hu J.J., Walker C., Ma X., Zhang Y., Chowdhury S., Meza-Sosa K.F., Lewandrowski M., Zhang H., Rowe M., McClelland A., Wu H., Junqueira C, & Lieberman J. (2023). The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells. Nature, 616(7956), 348-356.

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Rapid T-cell Activation Profiling

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TRAC-HIT and TRAC-CAR T cells were stained with Fixable Viability Dye eFluor 506 (eBioscience 65-0866-14) and rested for 30 minutes in standard tissue culture conditions. TRAC-HIT or TRAC-CAR T cells were incubated with Nalm6 target cells for 15 minutes at a TRAC-HIT/CAR T cell-to-target ratio of 1:2. After antigen stimulation, the cells were fixed with Phosflow Fix Buffer I (BD 557870). Fixed cells were stained for the HIT/CAR with AF647-GAM, followed by 2% mouse serum (15 min at 4 °C), and subsequently permeabilized with Phosflow Perm Buffer III (BD 558050) following the manufacturer’s procedure. Permeabilized samples were stained with antibodies detecting phosphorylated CD3ζ ITAM3 (pY142; BD 558448), ZAP70 (pY319; BD 557881) and ERK1/2 (pT202/pY204; BioLegend 369506).

Mansilla-Soto J., Eyquem J., Haubner S., Hamieh M., Feucht J., Paillon N., Zucchetti A.E., Li Z., Sjöstrand M., Lindenbergh P.L., Saetersmoen M., Dobrin A., Maurin M., Iyer A., Angus A.G., Miele M.M., Zhao Z., Giavridis T., van der Stegen S.J., Tamzalit F., Rivière I., Huse M., Hendrickson R.C., Hivroz C, & Sadelain M. (2022). HLA-independent T cell receptors for targeting tumors with low antigen density. Nature medicine, 28(2), 345-352.

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Quantifying Phosphorylated CD3ζ in CAR T Cells

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SFGγ−1928z, TRAC-1928z and TRAC-1XX T cells were fixed with Phosflow Fix Buffer I (BD) and stained for the CAR with GaM-af647 (Jackson ImmunoResearch), followed by 2% mouse serum, and subsequently permeabilized with Phosflow Perm Buffer III (BD) following the manufacturer’s procedure. Permeabilized samples were stained with antibodies detecting phosphorylated CD3ζ ITAM1 – af488 (EP776(2)Y; Abcam) or phosphorylated CD3ζ ITAM3 – PE (K25–407.69; BD).
All antibodies were titrated prior to use. Flow cytometric data were acquired on Fortessa X-20 (BD) or 5-laser Aurora (Cytek Biosciences) Flow cytometer voltages were calibrated with Ultra Rainbow Calibration Kit (SpheroTech, URCP-38–2K) prior to every acquisition. Analysis was performed using FCS Express 7 (De Novo Software). Negative and positive gates were set based on (un)stained PBMC and TiPS controls (Supplementary Fig. 1).

van der Stegen S.J., Lindenbergh P.L., Petrovic R.M., Xie H., Diop M.P., Alexeeva V., Shi Y., Mansilla-Soto J., Hamieh M., Eyquem J., Cabriolu A., Wang X., Abujarour R., Lee T., Clarke R., Valamehr B., Themeli M., Riviere I, & Sadelain M. (2022). Generation of T-cell-receptor-negative CD8αβ-positive CAR T cells from T cell-derived induced pluripotent stem cells. Nature biomedical engineering, 6(11), 1284-1297.

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STAT3 Phosphorylation in IL-6 Stimulated PBMCs

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The patient was hospitalized in the inpatient ward and treated with steroid, hydroxychloroquine and antimicrobial agents. Peripheral blood was collected and peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation. The cells were resuspended in RPMI 1640 supplemented with penicillin, streptomycin, and L-glutamine along with 10% fetal calf serum. PBMCs were then stimulated for 15 min with 20 ng/ml IL-6 (Peprotech) or not. Following stimulation, cells were immediately fixed with PhosFlow Fix Buffer I (BD Biosciences) for 10 min at 37°C, then permeabilized using BD Phosflow Perm Buffer III (BD Biosciences) following the manufacturer’s instruction. Cells were then stained with anti-STAT3 (pY705) or isotype-matched control antibodies (BD Biosciences) for 1 hour. Samples were collected with FACSCanto II (BD Biosciences) and analyzed with FlowJo Software (TreeStar).

Deng M., Li Y., Li Y., Mao X., Ke H., Liang W., Lei X., Lau Y.L, & Mao H. (2022). A Novel STAT3 Gain-of-Function Mutation in Fatal Infancy-Onset Interstitial Lung Disease. Frontiers in Immunology, 13, 866638.

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Phospho-STAT activation in MPN mice

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Protocol adapted from (Kalaitzidis and Neel, 2008 (link)). In brief, lineage-depleted bone marrow cells from 10–14-mo-old wild-type and Nol3^−/− MPN mice were incubated in IMDM 2% FBS for 30 min before cytokine stimulation. Cells were then stimulated with 50 ng/ml TPO, 10 ng/ml G-CSF, or PBS (unstimulated) for 15 min. After stimulation, cells were fixed using equal volume of Phosflow Fix Buffer I (BD) for 10 min at 37°C. Cells were pelleted by centrifugation for 5 min at 300 g, and after removal of supernatant, permeabilized using ice-cold acetone dropwise and incubated for 10 min on ice. Cells were then washed twice with 2% FBS in PBS, and stored in 2% FBS in PBS at 4°C until flow cytometry analysis. Samples were analyzed at the same time using a BD LSR II flow cytometer and antibodies against phospho-STAT5 (BD) and phospho-STAT3 (BD).

Stanley R.F., Piszczatowski R.T., Bartholdy B., Mitchell K., McKimpson W.M., Narayanagari S., Walter D., Todorova T.I., Hirsch C., Makishima H., Will B., McMahon C., Gritsman K., Maciejewski J.P., Kitsis R.N, & Steidl U. (2017). A myeloid tumor suppressor role for NOL3. The Journal of Experimental Medicine, 214(3), 753-771.

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Profiling T Cell Subsets by FACS

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Freshly isolated PBMCs were incubated with following fluorochrome-conjugated monoclonal antibodies in FACS buffer for surface staining. Antibodies (from BD Biosciences and BioLegend) were TCRαβ (clone IP26), CXCR3 (clone G025H7), CD45RA (clone HI100), CD3 (clone UCHT1), PD-1 (clone EH12.2H7), CD19 (clone SJ25C1), CCR6 (clone 11A9), CD8a (clone SK1), CCR4 (clone L291H4), CD62L (clone DREG-56), CD25 (clone BC96), CXCR5 (clone RF8B2), CD4 (clone RPA-T4), and dead cells stained with Zombie Aqua were excluded from analysis. After the surface staining, cells were washed with FACS buffer and then fixed with pre-warmed Phosflow™ Fix Buffer I (BD) in 37°C for 15 min. Cells were washed and resuspended in pre-chilled Phosflow Perm Buffer III (BD) in 4°C for 25 min. After wash, cells were stained with anti-pSTAT3 (BD, clone 4/pSTAT3) at 37°C for 30 min to detect pSTAT3 (pY705). The same procedure was performed to stain with an isotype control antibody (BD, clone G155-178). The expression of surface markers and intracellular pSTAT3 were analyzed by a FACS analyser (LSRFortessa X-20, BD). The results were analyzed with FlowJo software (TreeStar).

Deng J., Fan C., Gao X., Zeng Q., Guo R., Wei Y., Chen Z., Chen Y., Gong D., Feng J., Xia Y., Xiang S., Gong S., Yuan L., Shen W., Shen W., Lin L., Jiang T., He D., Lu L., Chen X, & Yu D. (2018). Signal Transducer and Activator of Transcription 3 Hyperactivation Associates With Follicular Helper T Cell Differentiation and Disease Activity in Rheumatoid Arthritis. Frontiers in Immunology, 9, 1226.

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Comprehensive Flow Cytometry Analysis of Immune Cells

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Cells were incubated with 0.5 µg/ml anti-CD16/32 (2.4G2, BD Bioscience) and Near IR Dead cell stain (Invitrogen) prior to staining with fluorescently labelled antibodies. Cells were fixed with Foxp3/Transcription Factor Buffer Staining Set (eBioscience) for between 30 min and 16 hr at 4°C.
For cytokine analysis, 10 µM Brefeldin A was added to cell cultures for 4 hr prior to staining for cell surface markers as described above. After overnight fixation, cells were permeabilized with Foxp3/Transcription Factor Buffer Staining Set (eBioscience) and were stained with intracellular markers or cytokines. Cells were analysed on a BD Fortessa or LSRII flow cytometer. Data were analysed using FlowJo (TreeStar). Antibodies are detailed in Table S1.
For pSTAT3 staining, inguinal, axillary and brachial LN cells were stained for surface markers, then stimulated with 100 ng/ml IL-23 (Biolegend) for 15 min. Cells were fixed with Phosflow Fix buffer I (BD Biosciences) at 37 °C for 10 min, before permeabilisation at 4 °C in Phosflow Perm Buffer III (BD Biosciences) for 30 min, staining with PE conjugated pSTAT3 (pY705) (BD Bioscience clone 4/P-STAT3) at room temperature for 30 min. Data were normalized to the mean stimulated WT data. ILCs were gated as live, single cell, CD45⁺, Lin^- (Ter119, F4/80, CD11b, CD11c, FceRIa, Gr1, CD19), CD3^-, TCRb^-, TCRgd^-, CD90.2⁺ CD127⁺.

Linley H., Ogden A., Jaigirdar S., Buckingham L., Cox J., Priestley M, & Saunders A. (2023). CD200R1 promotes interleukin-17 production by group 3 innate lymphoid cells by enhancing signal transducer and activator of transcription 3 activation. Mucosal Immunology, 16(2), 167-179.

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Phosphoflow and Cytokine Analysis

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For phosphoflow analysis, splenocytes were restimulated with plate-bound anti-CD3 (clone 500A2, BD Biosciences; coated using 1 µg/ml antibody in PBS) in a flat-bottomed 96 well plate for 30 min. Cells were then fixed and permeabilised using the BD Phosflow Fix Buffer I and Perm Buffer III according to the manufacturer’s instructions. Cells were stained with the appropriate surface and phosphoflow antibodies for 1 h at room temperature in PBS containing 2.5% Foetal Calf Serum and 0.1% Azide. For cytokine staining, cells were restimulated with 0.1 µg/ml of the appropriate synthesised viral peptide (Biomolecular Resource Facility, ANU) in the presence of 3 µg/ml Brefeldin A (eBioscience) and CD107a antibody for 6 h, prior to surface staining then fixation with Biolegend Fixation Buffer, and intracellular cytokine staining in eBioscience Permeabilisation Buffer according to manufacturer’s instructions.

Wagle M.V., Vervoort S.J., Kelly M.J., Van Der Byl W., Peters T.J., Martin B.P., Martelotto L.G., Nüssing S., Ramsbottom K.M., Torpy J.R., Knight D., Reading S., Thia K., Miosge L.A., Howard D.R., Gloury R., Gabriel S.S., Utzschneider D.T., Oliaro J., Powell J.D., Luciani F., Trapani J.A., Johnstone R.W., Kallies A., Goodnow C.C, & Parish I.A. (2021). Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance. Nature Communications, 12, 2782.

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PBMC Immunophenotyping and STAT5 Inhibition

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PBMCs were fixed with an equal volume of warm Phosflow Fix buffer I (BD) for 10 min at 37°C and then permeabilized for 10 min by cold Phosflow Perm/wash buffer III (BD) at 4°C, followed by surface CD4 and CD14, intracellular Foxp3, and STAT5 staining. For some experiments, STAT5 inhibitor (N′[(4-oxo-4H-chromen-3-yl)methylene] nicotinohydrazide) was added to PBMCs for 1 h before adalimumab treatment.

Nguyen D.X, & Ehrenstein M.R. (2016). Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF–TNF-RII binding in rheumatoid arthritis. The Journal of Experimental Medicine, 213(7), 1241-1253.

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