Phosflow lyse fix buffer 5x

Manufactured by BD

Phosflow Lyse/Fix Buffer 5X is a laboratory buffer solution used for cell preparation and analysis. It is a 5 times concentrated formulation that requires dilution before use. The buffer is designed to lyse cells and fix cellular proteins, preserving their phosphorylation state for subsequent analysis.

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Lab products found in correlation

Phosflow perm buffer 3, by BD (2 mentions) Pacific orange nhs ester, by Thermo Fisher Scientific (2 mentions) Dylight 350 nhs ester, by Thermo Fisher Scientific (2 mentions) Phosflow perm buffer 2, by BD (2 mentions) Vacutainer, by BD (1 mentions) Anti pakt pe clone rea359, by Miltenyi Biotec (1 mentions) Ic fixation buffer, by Thermo Fisher Scientific (1 mentions) Perm wash, by BD (1 mentions) Cbd500, by BD (1 mentions) Cd3 bv605 okt3, by BioLegend (1 mentions) Cd4 apc rpa t4, by BD (1 mentions) Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions) Cd8 pe cy5, by Beckman Coulter (1 mentions) Human interferon α, by Cell Signaling Technology (1 mentions) Recombinant human il 10, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum catalog no a3160601, by Thermo Fisher Scientific (1 mentions) Sodium azide catalog no s8032, by Merck Group (1 mentions)

Close spelling variants of this product

Lyse/fix Buffer 5X Phosflow Lyse/Fix BD Phosflow Fix Buffer Lyse/Fix Buffer Lyse/Fix Lyse buffer BD Phosflow

5 protocols using phosflow lyse fix buffer 5x

Detecting MM CTCs in Active Treatment

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Blood samples from three Stage III MM patients (on active treatment) were sourced from Conversant Bio (Huntsville, AL), collected in EDTA (BD Vacutainer®) tubes and shipped overnight to Epic Sciences for processing. Standard sample processing procedures were followed except blood was lysed using BD Phosflow™ Lyse/Fix Buffer 5X (BD Biosciences, San Diego, CA), and two slides from each patient were immunofluorescently labelled with an antibody cocktail against CD138, CD45, pS6 and stained with DAPI for MM CTC analysis. Following staining, CTCs were identified using Epic's proprietary algorithm as described above, and expression levels of CD138 and pS6 were measured.

Zhang L., Beasley S., Prigozhina N.L., Higgins R., Ikeda S., Lee F.Y., Marrinucci D, & Jia S. (2016). Detection and Characterization of Circulating Tumour Cells in Multiple Myeloma. Journal of Circulating Biomarkers, 5, 10.

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Phosphorylation Profiling of B-GCs

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Differentiated B-GCs without PRL were allowed to settle for 8 h in basal medium. Subsequently, the cells were incubated with PRL for 30 minutes. The cells were fixed with 1x BD Phosflow Lyse/Fix Buffer 5x (BD Biosciences)/10 minutes, with BD Phosflow Perm Buffer III (BD Biosciences); the cells were permeabilized to determine intracellular STAT1 (anti-pSTAT1 PE, clone A15158B, BioLegend), STAT3 (anti-pSTAT3 PE, clone 13A3-1, BioLegend), and STAT5 (anti-pSTAT5 PE, clone SRBCZX, eBioscience) phosphorylation. To determine AKT (anti-pAKT PE, clone REA359, Miltenyi Biotec) and ERK1/2 (anti-pERK1/2 PE, clone 6B8B69, BioLegend) phosphorylation, cells were treated with IC Fixation Buffer (eBioscience) for 30 minutes at 4°C, washed with FACS buffer or Perm/Wash (BD Biosciences), and incubated with the antibodies for flow cytometric analysis for 30 minutes at 4°C. The samples were read using a MACSQuant Analyzer 10 flow cytometer and the data were analyzed with FlowJo 10 software.

Carreón-Talavera R., Santana-Sánchez P., Fuentes-Pananá E.M., Legorreta-Haquet M.V., Chávez-Sánchez L., Gorocica-Rosete P.S, & Chávez-Rueda A.K. (2022). Prolactin promotes proliferation of germinal center B cells, formation of plasma cells, and elevated levels of IgG3 anti-dsDNA autoantibodies. Frontiers in Immunology, 13, 1017115.

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Multiparametric Flow Cytometry Panel

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The following FCB dyes were used: CBD500 (BD Biosciences, San Jose, CA, USA); Pacific Orange NHS ester, DyLight 350 NHS ester, and DyLight 800 NHS ester (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies tested for surface staining were: CD3-BV605 (OKT3) (BioLegend, San Diego, CA); CD4-APC (RPA-T4) (BD Biosciences, San Jose, CA, USA); CD8-PE-Cy5 (B9.11), and tube B of IOTest Beta Mark, containing Vβ 9-PE, Vβ 17-PE/FITC, and Vβ 16-FITC (FIN9, E17.5F3, and TAMAYA1.2) (Beckman Coulter, Miami, FL). LIVE/DEAD Fixable Aqua (a viability dye) for 405 nm excitation was used to exclude dead cells from analysis (Thermo Fisher Scientific). Aqua dye was dissolved in DMSO and stored at −80°C, according to the manufacturer’s instructions. Just before use, Aqua dye was diluted 1:16 with PBS and used for staining. All buffers (Phosflow Lyse/Fix Buffer 5X, Phosflow Perm Buffer II, and Phosflow Barcoding Wash Buffer 4X; BD Biosciences) were prepared, according to the manufacturer’s instructions.

Giudice V., Feng X., Kajigaya S., Young N.S, & Biancotto A. (2017). Optimization and Standardization of Fluorescent Cell Barcoding for Multiplexed Flow Cytometric Phenotyping. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 91(7), 694-703.

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Multi-Omics Immune Cell Analysis

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The following FCB dyes were used: DyLight 350 NHS ester and Pacific Orange NHS ester (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies used for surface staining were: mouse anti-human CD3-PerCP-Cy5.5 (clone SK7), mouse anti-human CD4-PE-Cy7 (clone SK3), mouse anti-human CD8-FITC (clone RPA-T8), and mouse anti-human CD20-APC-H7 (clone H1) (BD Biosciences, San Jose, CA, USA); and CD14-PE (clone M5E2) from BioLegend (San Diego, CA). Antibodies used for phosphoproteins were: pSTAT1(pY701)-Alexa Fluor 647 (clone 4a), pSTAT3(pY705)- Alexa Fluor 647 (clone 4/P-STAT3), and pSTAT5(pY694)- Alexa Fluor 647 (clone 47/Stat5 pY694) (BD Biosciences). Phosflow Lyse/Fix Buffer 5X, Phosflow Perm Buffer III, and Phosflow Barcoding Wash Buffer 4X buffers (BD Biosciences) were prepared and used according to manufacturer’s instructions. Phosflow Perm Buffer II (BD Biosciences) was diluted 1:1 with cold PBS and kept on ice before use. For PBMC stimulation, the following cytokines were used: recombinant human IL-10 (PeproTech, Rocky Hill, NJ); human Interferon-α (Cell Signaling Technology, Boston, MA); recombinant human IL-2 (Hoffmann-La Roche Inc, Nutley, NJ). The CTL Anti-Aggregate (CTL) wash buffer was from Cellular Technology Limited (Shaker Heights, OH).

Tsai W.L., Vian L., Giudice V., Kieltyka J., Liu C., Fonseca V., Gazaniga N., Gao S., Kajigaya S., Young N.S., Biancotto A, & Gadina M. (2019). High Throughput pSTAT Signaling Profiling by Fluorescent Cell Barcoding and Computational Analysis. Journal of immunological methods, 477, 112667.

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Phosflow Lyse/Fix Buffer Kinase Assay

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Phosflow Lyse/Fix Buffer 5X (Catalog No. 558049) was purchased from BD Biosciences. Fetal bovine serum (Catalog No. A3160601) was purchased from Thermo Fisher Scientific and sodium azide (Catalog No. S8032) was obtained from Sigma Aldrich. D‐PBS (without Ca²⁺ or Mg²⁺) was obtained from Invitrogen (Catalog No. 14190). Glutathione S‐transferase (GST)‐tagged recombinant human kinase domains of JAK1, JAK2, and JAK3 were purchased from Thermo Fisher Scientific. His‐tagged recombinant human TYK2 kinase domain was expressed in SF21/baculovirus and purified using a two‐step affinity (Ni‐nitrilotriacetic acid) and size‐exclusion (SEC S200) purification method.

Dowty M.E., Lin T.H., Jesson M.I., Hegen M., Martin D.A., Katkade V., Menon S, & Telliez J. (2019). Janus kinase inhibitors for the treatment of rheumatoid arthritis demonstrate similar profiles of in vitro cytokine receptor inhibition. Pharmacology Research & Perspectives, 7(6), e00537.

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