Cd27 pe cy7

Manufactured by BD

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CD27-PE-Cy7 is a fluorescently labeled antibody that binds to the CD27 antigen. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on T cells, B cells, and natural killer cells. The PE-Cy7 fluorescent label allows for the detection and analysis of CD27-positive cells using flow cytometry.

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CD27-PE (41 citations) Anti-CD27-PE-Cy7 (11 citations) CD27 PE-Cy7-clone M-T271 (2 citations)

20 protocols using cd27 pe cy7

Multivariate Flow Cytometry Analysis of Immune Cell Subsets

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Organoids were harvested from the upper portion of the permeable membranes by rinsing the membranes with PBS. Cells were washed with FACS buffer (PBS + 0.1% BSA, 0.05% sodium azide and 2 mM EDTA) and stained at 4 °C with the following anti-human antibodies in the presence of Fc block and live/dead Aqua Zombie stain, all from BioLegend unless otherwise noted: FITC CD138 (1/100), FITC CD116 (1/100), FITC CD21 (1/50), FITC or Ax488 CXCR5 (1/33), PerCP-Cy5.5 CD8 (1/100), PerCP-Cy5.5 CD33 (1/100), PE CD19 (1/100), PE CD56 (1/100), PE gamma-delta TCR (1/100), PE-Cy7 CD27 (1/100), PE-Cy7 CD123 (1/50), PE-Cy7 CD8 (1/100), APC CD38 (1/200), APC HLA-DR (1/100), APC CD27 (1/200), Ax700 CD45 (1/100), Ax700 CD14 (1/100), APC-Cy7 IgD (1/50), APC-Cy7 CD16 (1/100), APC-Cy7 CD45RA (1/100), Pacific Blue HLA-DR (1/100), Pacific Blue PD-1 (1/50), BV605 CD3 (1/100), BV650 CD4 (1/100), BV650 CD19 (1/100), BUV395 IgM (1/20; BD Biosciences) and BUV395 CD45RA (1/20; BD Biosciences).
For AID staining, after surface staining, the cells were fixed and permeabilized (eBioscience) and stained intracellularly with biotinylated anti-AID antibody (1/100; clone mAID-2; eBioscience) followed by PE-streptavidin (eBioscience). A no-AID antibody control was used to discriminate positive signal. All analyzer data were collected on BD LSRII instruments and analyzed using FlowJo (TreeStar).

Wagar L.E., Salahudeen A., Constantz C.M., Wendel B.S., Lyons M.M., Mallajosyula V., Jatt L.P., Adamska J.Z., Blum L.K., Gupta N., Jackson K.J., Yang F., Röltgen K., Roskin K.M., Blaine K.M., Meister K.D., Ahmad I.N., Cortese M., Dora E.G., Tucker S.N., Sperling A.I., Jain A., Davies D.H., Felgner P.L., Hammer G.B., Kim P.S., Robinson W.H., Boyd S.D., Kuo C.J, & Davis M.M. (2021). Modeling human adaptive immune responses with tonsil organoids. Nature medicine, 27(1), 125-135.

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Immune Profiling of COVID-19 Patients

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Left-over blood samples for clinical examination from 32 COVID-19 patients and 37 healthy volunteers were collected for analysis. All the samples were processed in the Clinical Lab of Wuhan Union Hospital based on uniform standard procedure. Briefly, peripheral blood mononuclear cells (PBMCs) and plasma were isolated by density gradient centrifugation. Fresh separated PBMCs were stained for flow cytometry, and plasma samples were used for cytokine detection.
Flow cytometry was performed as described previously (9 (link)). Briefly, 1 × 10⁶ PBMCs were stained with indicated antibodies in the dark at room temperature for 20 min. After several washes, the cells were analyzed within 1 h. All samples were detected by BD FACS Canto II Flow Cytometry System and analyzed with the BD FACS Diva Software. Antibodies used for flow cytometry included FITC-CD3 (clone: HIT3a), PE-PD-1 (clone: EH12.1), PE/Cy7-CD56 (clone: B159), APC-CD244 (clone: 2-69), APC/Cy7-CD45 (clone: 2D1), BV421-CD16 (clone: 3G8), BV421-CD4 (clone: RPA-T4), PerCP-CD8 (clone: RPA-T8), PE/Cy7-CD27 (clone: M-T271), and all of these were purchased from BD Pharmingen.

Li M., Guo W., Dong Y., Wang X., Dai D., Liu X., Wu Y., Li M., Zhang W., Zhou H., Zhang Z., Lin L., Kang Z., Yu T., Tian C., Qin R., Gui Y., Jiang F., Fan H., Heissmeyer V., Sarapultsev A., Wang L., Luo S, & Hu D. (2020). Elevated Exhaustion Levels of NK and CD8+ T Cells as Indicators for Progression and Prognosis of COVID-19 Disease. Frontiers in Immunology, 11, 580237.

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Multiparameter Flow Cytometry Analysis

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After 6 days culture, cultured cells were analyzed for expression of surface markers by multi-color flow cytometry. Cell aliquots were treated with anti-human Fc mAb for 20 minutes and stained for 30 minutes with selected combinations of fluorochrome-conjugated antibodies.
For the analysis of B cell subpopulations and activation markers, the monoclonal antibodies used were CD19-PE-CF594 (BD Biosciences, San Jose, CA), CD62L-PE-Cy5 (BD Biosciences), IgD-FITC (BD Biosciences), CD27-PE-Cy7 (BD Biosciences), CD24-PE (BD Biosciences), CD38-PerCP-Cy5.5 (Beckman Coulter, Brea CA), CD138-PE-Cy7 (eBioscience, San Diego, CA), CD23-PE (BD Biosciences), CD21-PE-Cy5 (BD Biosciences), IgG-PC7 (BD Biosciences), CD86-Alexa 700 (BD Biosciences) and CD95-Pacific Blue (BioLegend, San Diego, CA).
For plasma cell staining, cultured cells were first incubated with monoclonal antibodies to surface markers (CD38, CD138 and CD19), fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) and then incubated with Blimp-1-PE (R&D systems) and Pax5-APC (eBioscience) for 30 minutes.
For the proliferation assay, 1–10×10⁶ cells of interest were labeled with 1μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) before culture initiation. After 6 days of culture, cells were harvested, incubated with antibodies, and dilution of CFSE was assessed by flow cytometry.

Traitanon O., Mathew J.M., La Monica G., Xu L., Mas V, & Gallon L. (2015). Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells. PLoS ONE, 10(6), e0129658.

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Comprehensive Immune Cell Profiling

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The monoclonal antibodies used in this study included CD3 PerCP, CD5 APC-R700, CD8-FITC, CD11c PE-Cy5, CD14 APC-H7, CD19 PE-Cy5, CD25 PE-Cy7, CD24 FITC, CD27 PE-Cy7, CD28 PE-Cy5, CD38 APC-H7, CD45 V500, CD45RO APC, CD56 APC, CD57 FITC, CD80 PE, CD86 APC, CD123 APC, CD127 BV421, CCR7 PE, TIGIT BV421, HLA DR V450, TIM3 PE, CXCR5 APC-R700, PD1 BV421, and PDL1 PE-Cy7 (BD Biosciences).

Rodero-Romero A., Sainz de la Maza S., Fernández-Velasco J.I., Monreal E., Walo-Delgado P.E., Chico-García J.L., Villarrubia N., Rodríguez-Jorge F., Rodríguez-Ramos R., Masjuan J., Costa-Frossard L, & Villar L.M. (2023). Blood CD8+ Naïve T-Cells Identify MS Patients with High Probability of Optimal Cellular Response to SARS-CoV-2 Vaccine. Vaccines, 11(9), 1399.

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Multiparameter Immune Cell Analysis

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CD3-Alexa Fluor 700, CD4-BV605, CD8-APCy7, CD38-APC, CD27-PE-Cy7, CD19-PE-TxR, PD1-PE, ICOS-PcPCy5.5 and CXCR5-AF488 (BD Biosciences).

Currenti J., Simmons J., Oakes J., Gaudieri S., Warren C.M., Gangula R., Alves E., Ram R., Leary S., Armitage J.D., Smith R.M., Chopra A., Halasa N.B., Pilkinton M.A, & Kalams S.A. (2023). Tracking of activated cTfh cells following sequential influenza vaccinations reveals transcriptional profile of clonotypes driving a vaccine-induced immune response. Frontiers in Immunology, 14, 1133781.

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Isolation and Characterization of PBMCs

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Peripheral blood mononuclear cells (PBMCs) were obtained from healthy individuals homozygous for the FcγRIIB-T232 or FcγRIIB-I232 site, under appropriate ethics approval from the NIHR Cambridge Bioresource. Inclusion criteria for individuals were people aged between 44 and 77 years, with no serious co-morbidities, no direct family history of autoimmune disease, no use of immunosuppressants or steroids and no hospitalisation within the last 12 months. Individuals were age and sex matched (18 females and 11 males matched between genotypes). Flow sorting was performed using CD19-BV785, CD38-BV711, CD3-NC650, CD14-605NC, CD24-PerCP-Cy5.5, IgD-FITC, CD27-PE-Cy7 (all from BD Bioscience) and Aqua (for live-dead cell detection, Invitrogen), where flow protocol is outlined in Supplementary Fig. 7.

Espéli M., Bashford-Rogers R., Sowerby J.M., Alouche N., Wong L., Denton A.E., Linterman M.A, & Smith K.G. (2019). FcγRIIb differentially regulates pre-immune and germinal center B cell tolerance in mouse and human. Nature Communications, 10, 1970.

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Multiparameter Flow Cytometry Staining

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The following antibodies were used for cell surface staining: CD3-eFluor 780-allophycocyanin (eF780-APC; clone UCHT1), TCR-allophycocyanin (APC; clone IP26), and IL-2–APC (clone MQ1-17H12) (all from eBioscience); CD4-BD Horizon R phycoerythrin-CF594 (PE-CF594; clone RPA-T4), CD27-PE-Cy7 (Pe-Cy7-clone M-T271), CXCR5-Alexa Fluor 488 (AF488; clone RF8B2), and CCR7-Alexa Fluor 647 (AF647; clone 3D12) (all from BD Biosciences); CD14-Viogreen (clone TÜK4) and CD20-Viogreen (clone LT29) (from Miltenyi Biotec); and CD38-Alexa Fluor 700 (AF700; clone HIT2), CD20-peridinin chlorophyll protein (PerCP)/Cy5.5 (clone 2H7), CD8-brilliant violet 785 (BV785; clone RPA-T8), CD45RA-brilliant violet 421 (BV421; clone HI100), CCR7-PE-Cy7 (clone G043H7), and CD3-APC (clone SK7) (all from BioLegend). The fixable viability dye eFluor 506 (eF506; eBioscience) was added to restrict the analysis to live cells.

Claireaux M., Galperin M., Benati D., Nouël A., Mukhopadhyay M., Klingler J., de Truchis P., Zucman D., Hendou S., Boufassa F., Moog C., Lambotte O, & Chakrabarti L.A. (2018). A High Frequency of HIV-Specific Circulating Follicular Helper T Cells Is Associated with Preserved Memory B Cell Responses in HIV Controllers. mBio, 9(3), e00317-18.

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Comprehensive Immune Cell Profiling

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The following monoclonal antibodies were purchased from eBioscience: CD278 (ICOS)-biotin, CD27-PeCy7, Foxp3-FITC, RORγt-PE, T-bet-PE; or from BD Biosciences: PD1-PECF594, CXCR3-APC, CD24-PECF594, CD25-BB515, CD44-PECy7, CD4-A700, CD8-A700, CD4-PB, CD62L-A700, GATA3-PE, RORγt-PECF594, STAT1 (pY701)-A488, IFNγ-PE, IL-10-APC, IL-17-PerCP-Cy5.5, streptavidin-PECy7. Live/dead fixable near-IR stain (Thermo Fisher) was used to exclude dead cells. For transcription factor staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer’s protocol (Foxp3 staining buffer set from eBioscience). For cytokine staining, cells were stimulated in media containing phorbol 12-myristate 13-acetate (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml, Sigma-Aldrich), and brefeldin-A (1/100, eBioscience) for 3 hr. After stimulation, cells were stained for surface markers, followed by fixation and permeabilization before intracellular staining according to the manufacturer’s protocol (cytokine staining buffer set from BD Biosciences). For phosphorylation staining, cells were stimulated with IFN-γ (50 ng/ml, PeproTech) for 30 min, fixed with formaldehyde, and permeabilized with methanol before staining. Flow cytometric analysis was performed on a Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Ajouaou Y., Azouz A., Taquin A., Denanglaire S., Hussein H., Krayem M., Andris F., Moser M., Goriely S, & Leo O. (2022). The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells. eLife, 11, e70555.

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Multiparametric Flow Cytometry Immunophenotyping

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Cell surface staining was processed using whole blood lysis method. Freshly drawn heparinized peripheral blood samples were labeled with monoclonal antibody (mAb) panel for T lymphocyte subsets [anti-CD3-AlexaFlour700, -CD4-APC, -CD8-APC/CY7, -CD45-BV510, -CD45RA-FITC, -CD45RO-PE, -CCR7-PE and -HLA-DR-BV711 (all from Biolegend, San Jose, CA, USA)], mAb panel for B lymphocyte subsets [anti-CD19-APC, -CD24-PE, -CD27-PE/CY7, -CD38-AlexaFlour700, -CD45-BV510, -CD138-BV421 and -IgD-FITC (all from BD-Biosciences, San Jose, CA, USA)], and with mAb panel for Treg [anti-CD3-AlexaFlour700, -CD4-APC, -CD25-PE, -CD45-BV510, -CD127-BV421 (all from BD-Biosciences, San Jose, CA, USA)]. Following incubation, erythrocytes were lysed using FACS Lysing Solution (BD Biosciences, San Jose, CA, USA), and at least 10.000 cells were collected in CD45⁺ lymphocyte gate. The data were acquired on a NovoCyte flow cytometer (Agilent Technologies, USA) and analyzed using the NovoExpress operating system software (Agilent Technologies, USA).

Gelmez M.Y., Oktelik F.B., Tahrali I., Yilmaz V., Kucuksezer U.C., Akdeniz N., Cetin E.A., Kose M., Cinar C., Oguz F.S., Besisik S., Koksalan K., Ozdemir O., Senkal N., Gul A., Tuzun E, & Deniz G. (2022). Immune modulation as a consequence of SARS-CoV-2 infection. Frontiers in Immunology, 13, 954391.

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Isolation of SARS-CoV-2 RBD-Specific B Cells

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RBD-specific single B cells were sorted as previously described [29 (link)]. In brief, PBMCs from infected individuals were collected and incubated with an antibody cocktail and a His-tagged RBD protein for identification of RBD-specific B cells. The cocktail consisted of the Zombie viability dye (Biolegend), CD19-Percp-Cy5.5, CD3-Pacific Blue, CD14-Pacific Blue, CD56-Pacific Blue, IgM-Pacific Blue, IgD-Pacific Blue, IgG-PE, CD27-PE-Cy7 (BD Biosciences) and the recombinant RBD-His described above. Two consecutive staining steps were conducted: the first one used an antibody and RBD cocktail incubation of 30 min at 4 °C; the second staining involved staining with anti-His-APC and anti-His-FITC antibodies (Abcam) at 4 °C for 30 min to detect the His tag of the RBD. The stained cells were washed and resuspended in PBS containing 2% FBS before being strained through a 70-μm cell mesh filter (BD Biosciences). RBD-specific single B cells were gated as CD19 ⁺CD27 ⁺CD3^-CD14^-CD56^-IgM^-IgD^-IgG ⁺RBD⁺ and sorted into 96-well PCR plates containing 10 μL of RNAase-inhibiting RT–PCR catch buffer (1M Tris-HCl pH 8.0, RNase inhibitor, DEPC-treated water). Plates were then snap-frozen on dry ice and stored at −80 °C until the reverse transcription reaction.

Zhou B., Zhou R., Chan J.F., Zeng J., Zhang Q., Yuan S., Liu L., Robinot R., Shan S., Liu N., Ge J., Kwong H.Y., Zhou D., Xu H., Chan C.C., Poon V.K., Chu H., Yue M., Kwan K.Y., Chan C.Y., Chan C.C., Chik K.K., Du Z., Au K.K., Huang H., Man H.O., Cao J., Li C., Wang Z., Zhou J., Song Y., Yeung M.L., To K.K., Ho D.D., Chakrabarti L.A., Wang X., Zhang L., Yuen K.Y, & Chen Z. (2023). SARS-CoV-2 hijacks neutralizing dimeric IgA for nasal infection and injury in Syrian hamsters1. Emerging Microbes & Infections, 12(2), 2245921.

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