Phosphoflow fix buffer 1

Manufactured by BD

Phosphoflow Fix Buffer I is a laboratory reagent used for the fixation and permeabilization of cells prior to flow cytometry analysis. It is designed to preserve the phosphorylation state of cellular proteins, enabling the detection and quantification of phosphorylated protein targets.

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Lab products found in correlation

Phosphoflow perm buffer 3, by BD (3 mentions) Anti stat 5p af647 antibody clone 47 py694, by BD (3 mentions) Ifn γ clone 4s b3, by BD (1 mentions) Pstat 4 clone 38, by BD (1 mentions) Fitc labeled goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions) Stat1, by Cell Signaling Technology (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Perm wash buffer 1, by BD (1 mentions) Mitotracker green, by Cell Signaling Technology (1 mentions) Fix and perm cell permeabilization kit, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions) Perm buffer 3, by BD (1 mentions)

Close spelling variants of this product

Fix Buffer I BD Phosphoflow

6 protocols using phosphoflow fix buffer 1

Flow Cytometry Analysis of Human Cells

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The following antibodies were used to perform flow cytometry on human cells (all antibodies are from Biolegend unless indicated otherwise): ARG1 (clone 14D2C43), CCR7 (clone G043H7), anti-CD3 (clone RPA-T4), CD4 (clone A161A1), CD8 (clone SK1), CD45RA (clone H1100), CD45RO (clone UCHL1), IFN-γ (clone 4S.B3), IL-10 (clone JES-9D7), IL-4 (clone MP4–25D2), IL-17A (clone BL168), TNFa (Mab11), Ki67 (clone B56, BD biosciences), pSTAT-4 (clone 38, BD Biosciences), pSTAT-1 (clone 4a, BD Biosciences).
Cell surface CAT-1 detection was performed using a soluble ligand derived from the receptor-binding domain (RBD) of the bovine leukemia virus (BLV) envelope glycoprotein (BLV-RBD).^{66 (link)} Staining was realized with a rabbit Fc-tagged BLV-RBD (BLV.RBD.rabbitIgG1-Fc) (Metafora-biosystems), followed by an anti-rabbit IgG1-Fc secondary antibody (Thermo Fisher), as previously described.^{66 (link)}For intracellular staining, BDcytofix/cytoperm buffers were used, except for FOXP3 staining (#00–5523-00, Thermo Fisher) and for pSTAT-1 and pSTAT-4 (# 557870, BD Biosciences phosphoflow fix buffer I and #554656, BD Biosciences phosphoflow perm buffer III) were used according to manufacturer’s protocol.

West E.E., Merle N.S., Kamiński M.M., Palacios G., Kumar D., Wang L., Bibby J.A., Overdahl K., Jarmusch A.K., Freeley S., Lee D.Y., Thompson J.W., Yu Z.X., Taylor N., Sitbon M., Green D.R., Bohrer A., Mayer-Barber K.D., Afzali B., Kazemian M., Scholl-Buergi S., Karall D., Huemer M, & Kemper C. (2023). Loss of CD4+ T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection. Immunity, 56(9), 2036-2053.e12.

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Quantification of STAT-5 Phosphorylation

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After 3 days of in vitro activation the cells were harvested and washed multiple times to remove endogenous IL-2. The cells were treated with increasing doses of muPD-1-IL-2v or muFAP-IL-2v for 30min at 37°C. Directly after treatment, the cells were fixed with Phosphoflow Fix Buffer I (BD) and incubated for 30 min at 37°C. The cells were then permeabilized overnight at −80°C with Phosphoflow PermBuffer III (BD) before being stained for 30min at 4°C with anti-STAT-5P-AF647 antibody (clone 47/pY694 (1:20), BD Biosciences).

Brune D, & Kim S. (1993). Predicting protein diffusion coefficients.. Proceedings of the National Academy of Sciences of the United States of America, 90(9), 3835-3839.

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Quantification of STAT-5 Phosphorylation

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Brune D, & Kim S. (1993). Predicting protein diffusion coefficients.. Proceedings of the National Academy of Sciences of the United States of America, 90(9), 3835-3839.

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Evaluation of PD1-IL2v Fusion Proteins

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After 3 d of in vitro activation, cells were collected and washed multiple times to remove endogenous IL-2. A portion of the CFSE-labelled T cells were exposed to 10 µg ml^–1 of parental anti-PD-1 antibody to block the PD-1 epitope for 30 min at room temperature and, thereafter, unbound antibody was washed away.
To assess IL-2R signalling (STAT5-P) on human T cells following treatment, both anti-PD-1-pretreated and untreated cells were exposed to increasing concentrations of PD1-IL2v, FAP-IL2v or FAP-IL2 superkine analogue^{24 (link)} for 12 min at 37 °C. To investigate the cis/trans binding of PD1-IL2v, anti-PD-1-pretreated or untreated CFSE-labelled cells were co-cultured 1:1 with untreated CTV-labelled cells. Cells were then exposed for 12 min at 37 °C to 0.1 μg ml^–1 (630 pM) of the treatment fusion proteins.
For the mouse ex vivo experiment, the cells were treated with increasing doses of muPD1-IL2v or muFAP-IL2v for 30 min at 37 °C.
Directly after treatment, cells were fixed with Phosphoflow Fix Buffer I (BD) and incubated for 30 min at 37 °C. Cells were then permeabilized overnight at −80 °C with Phosphoflow PermBuffer III (BD) before being stained for 30 min at 4 °C with anti-STAT5-P–AF647 antibody (clone 47/pY694, BD Biosciences; 1:20).

Codarri Deak L., Nicolini V., Hashimoto M., Karagianni M., Schwalie P.C., Lauener L., Varypataki E.M., Richard M., Bommer E., Sam J., Joller S., Perro M., Cremasco F., Kunz L., Yanguez E., Hüsser T., Schlenker R., Mariani M., Tosevski V., Herter S., Bacac M., Waldhauer I., Colombetti S., Gueripel X., Wullschleger S., Tichet M., Hanahan D., Kissick H.T., Leclair S., Freimoser-Grundschober A., Seeber S., Teichgräber V., Ahmed R., Klein C, & Umaña P. (2022). PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells. Nature, 610(7930), 161-172.

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Quantification of IFNγ Receptor and SOCS3

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Monocytes were identified using HLA-DR, CD14 and CD16, as described above. Cell surface expression of the IFNγ receptor R1 was determined with IFNγR1 antibody (CD119, R&D Systems, Minneapolis, MN). For intracellular detection of SOCS3, cells were permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), stained for SOCS3 (Abcam, Cambridge, MA), followed by APC-labeled goat anti-rabbit antibody (R&D Systems). For intracellular detection of total STAT1, BD PhosphoFlow Fix Buffer I and Perm/Wash Buffer I were used. Cells were stained for STAT1 (Cell Signaling Technologies, Beverly, MA), followed by FITC-labeled goat anti-mouse antibody (Invitrogen, Carlsbad, CA).

Macaubas C., Wong E., Zhang Y., Nguyen K.D., Lee J., Milojevic D., Shenoi S., Stevens A.M., Ilowite N., Saper V., Lee T, & Mellins E.D. (2015). Altered signaling in systemic juvenile idiopathic arthritis monocytes. Clinical immunology (Orlando, Fla.), 163, 66-74.

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T Cell Immunophenotyping and Signaling

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T cells were washed in FACS buffer (DPBS no calcium, no magnesium
(Gibco) with 2% FBS). Cells were incubated on ice in FACS buffer with antibodies
specific for cell surface markers for twenty minutes. Antibodies used are in
Table S4. For
pSTAT5 staining: cells were prepared with the Fix and Perm Cell Permeabilization
kit (ThermoFisher) according to manufacturer’s instructions. For pS6
staining: T cells were cultured 24 hours in complete AIM-V media without IL-2
prior anti-idiotype stimulation (1ug/ml of mouse IgG anti-human CD19 idiotype
crosslinked with 10ug/ml of anti-mouse Fab in PBS). Cells were incubated with
anti-idiotype - or left unstimulated - for 1 hour at 37C and kept on ice
immediately afterwards. Cells were fixed (BD Phosphoflow Fix Buffer I) and then
permeabilized (BD Perm Buffer III) following manufacturer’s instructions.
For mitochondrial mass staining: cells were stained with MitoTracker Green (Cell
Signaling Technology) at 200 nM, 37°C for 30 minutes. Prior to
acquisition, cells resuspended in FACS buffer and analyzed on a LSRFortessa (BD)
with BD FACSDiva software.

Freitas K.A., Belk J.A., Sotillo E., Quinn P.J., Ramello M.C., Malipatlolla M., Daniel B., Sandor K., Klysz D., Bjelajac J., Xu P., Burdsall K.A., Tieu V., Duong V.T., Donovan M.G., Weber E.W., Chang H.Y., Majzner R.G., Espinosa J.M., Satpathy A.T, & Mackall C.L. (2022). Enhanced T Cell Effector Activity by Targeting Mediator Kinase Module. Science (New York, N.Y.), 378(6620), eabn5647.

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