Anti cd27 apc cy7

Manufactured by BioLegend

Anti-CD27-APC-Cy7 is a fluorochrome-conjugated monoclonal antibody that binds to the CD27 cell surface receptor. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on various immune cell types. This reagent can be used for the identification and characterization of CD27-expressing cells in flow cytometric analysis.

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Lab products found in correlation

Anti cd45ro fitc, by BD (2 mentions) Anti cd3 alexa 700, by BD (2 mentions) Facsdiva software, by BD (2 mentions) Anti cd24 fitc, by BD (1 mentions) Anti cd38 pe, by Beckman Coulter (1 mentions) Facsverse flow cytometer, by BD (1 mentions) Anti cd3 pe cy7, by BioLegend (1 mentions) Anti cd28 apc, by BioLegend (1 mentions) Anti cd4 percp, by BD (1 mentions) Anti cd8 pe, by BD (1 mentions) Facscanto 2 cytometer, by BD (1 mentions) Anti igd fitc, by BD (1 mentions) Perm wash, by BD (1 mentions) Lsrii machine, by BD (1 mentions) Cd38 pe cy7, by BioLegend (1 mentions) Igd apc cy7, by BioLegend (1 mentions) Cd21 pe cy7, by BioLegend (1 mentions) Cd138 apc, by BioLegend (1 mentions) Cd43 pe, by BioLegend (1 mentions) Cd5 fitc, by BioLegend (1 mentions)

8 protocols using anti cd27 apc cy7

Quantification of CD19+ B Cells

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CD19+ B cell counts were determined on whole blood after red blood cell lysis using mAbs specific for CD19 (BD Bioscience, San Jose, CA). Absolute numbers of B cells were calculated based on patient white blood count and the immunophenotypic data. Phenotyping analysis of B cells was performed by labeling blood lymphocytes with titrated volumes of anti-CD24 FITC (BD biosciences), anti-CD38 PE (Beckman Coulter, Fullerton, CA), anti-CD27APC/Cy7 (Biolegend, San Diego, CA). Cells were analyzed using a FACSVerse flow cytometer (BD Biosciences). The flow cytometer calibrated by methods described previously^{12 (link)}.

Gao B., Gu Y., Rong C., Moore C., Porcheray F., Wong W., Preffer F., Saidman S.L., Fu Y., Cosimi B., Sachs D.H., Kawai T., Sykes M, & Zorn E. (2017). Dynamics of B cell recovery following kidney/bone marrow transplant recipients. Transplantation, 101(11), 2722-2730.

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Multiparametric Immune Cell Profiling

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PBMC were washed with PBS and stained with anti-CD3-PE-Cy7 (Biolegend), anti-CD4-PerCP (BD Pharmingen), anti-CD8-PE (BD Pharmingen), anti CD28-APC (Biolegend), anti CD45RO-FITC (BD Pharmingen), anti-CD20-PerCP (Biolegend), anti-CD27-APC-Cy7(Biolegend) and anti-IgD-FITC (BD Pharmingen) antibodies for 20 min, 4 °C in the dark. After washing with PBS, cells were analyzed using a FACS Canto II cytometer and FACSDiva software (BD).

Weinberger B., Keller M, & Grubeck-Loebenstein B. (2017). Long-term maintenance of diphtheria-specific antibodies after booster vaccination is hampered by latent infection with Cytomegalovirus. Immunity & Ageing : I & A, 14, 16.

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Multiparametric Flow Cytometry Analysis of B Cell Subsets

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Cells were stained with panels of antibodies including anti-mouse CD19-pacific Blue, CD138-APC, CD21-PEcy7, CD5-FITC, CD43-PE, IgD-APCcy7, CD38-PE-Cy7, GL-7-Percp (BioLegend, San Diego, CA ), and IgM-APCcy7 (Miltenyi Biotec, Auburn, CA). For the human B cell phenotypes, the following antibodies were used: anti-CD19-pacific blue, anti-CD38-PECy7, anti- CD20-PE, anti-CD27-APCcy7 (BioLegend), and anti– human IgG-PE, anti– human IgA-APC (Miltenyi Biotec). BD Cytofix/ Cytoperm and BD Perm/Wash (BD) were used for IgA and IgG intracellular staining per manufacturers’ instructions. Data were analyzed on a BD Biosciences LSRII machine and Flowjo software 29.

Qu P., Wuest T., Min Y., Alevizos I., Young H.A, & Lin P.C. (2020). Natural Killer Cell Transcript 4 promotes the development of Sjögren’s Syndrome via activation of Rap1 on B cells. Journal of autoimmunity, 116, 102559.

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Isolation and Sorting of Tfh Cells and Naive B Cells

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PBMCs were separated by gradient centrifugation with a Lymphoprep (Axis-Shield; Oslo, Norway) according to the manufacturer’s instructions. CD4⁺ T cells and CD19⁺ B cells were enriched from PBMCs using a CD4⁺ T cell isolation kit and CD19⁺ B cell isolation kit, respectively (Miltenyi Biotec; Bergisch Gladbach, Germany) according to the manufacturer’s instructions. For sorting of Tfh cell subsets, enriched CD4⁺ T cells were stained with anti-CCR6-PE (11A9, BD Biosciences), anti-CXCR3-APC (1C6, BD Biosciences), anti-CXCR5-PerCP/Cy5.5 (J252D4, BioLegend), anti-CD45RA-BV421 (HI-100, BioLegend), and anti-CD4-HorizonV500 (RPA-T4, BD Biosciences). Tfh cell subsets were sorted from CD4⁺ T cells as Tfh2 (CXCR3^-CCR6^-), Tfh1 (CXCR3⁺CCR6^-), or Tfh17 (CXCR3^-CCR6⁺) cells among CXCR5⁺CD45RA^-CD4⁺ T cells [19 (link)]. For sorting of naïve B cells, enriched CD19⁺ B cells were stained with anti-IgD-PE (IA6-1, Biolegend) and anti-CD27-APC/Cy7 (O323, BioLegend). Naïve B cells were sorted from CD19⁺ B cells as IgD⁺CD27^-CD19⁺ cells. Tfh cell subsets and naïve B cell sorting was conducted with a FACS Aria III (BD Biosciences) according to the manufacturer’s instructions. Cell purity of naïve B cells determined by flow cytometry was greater than 98 % and cell purity was greater than 93 % for Tfh cell subsets.

Akiyama M., Yasuoka H., Yamaoka K., Suzuki K., Kaneko Y., Kondo H., Kassai Y., Koga K., Miyazaki T., Morita R., Yoshimura A, & Takeuchi T. (2016). Enhanced IgG4 production by follicular helper 2 T cells and the involvement of follicular helper 1 T cells in the pathogenesis of IgG4-related disease. Arthritis Research & Therapy, 18, 167.

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Isolation and Sorting of SARS-CoV-2 S-Specific Memory B Cells

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B cells, enriched from PBMCs with human CD19 MicroBeads (Miltenyi), were incubated with 2 μg/ml flag-tagged S protein or mixture of flag-tagged S protein (Genscript, Cat. Z03481) and His-tagged RBD (Chen et al., 2020 ) on ice for 30 minutes. Cells were then washed with 2% fetal bovine serum (FBS) (Hyclone) in PBS and stained with mixture of anti-human IgG (Percpcy5.5; Biolegend Cat. 410710), anti-human IgD (FITC; Biolegend Cat. 348205), anti-human IgM (Bv605; Biolegend Cat. 314524), anti-CD27 (APCcy7; Biolegend Cat. 356404). A mixture of PE- and APC-conjugated anti-flag antibodies (Biolegend Cat. 637309 and 637307) was also added for gating S-specific double positive cells, or a mixture of PE-conjugated anti-His (Biolegend Cat. 362603) and APC-conjugated anti-flag, for S positive and RBD negative cells. Memory B-cells were gated on DAPI⁻CD19⁺IgM⁻IgD⁻IgG⁺CD27⁺. Individual S double positive or S+RBD- cells were sorted with a FACSAria Fusion (BD Biosciences) into each well of 96-well microplates containing 4 μl/well of lysis buffer (0.5X PBS, 10 mM dithiothreitol, and 4U RNaseOUT). Lysed cells were immediately frozen and stored at −80 °C until use.

Tong P., Gautam A., Windsor I.W., Travers M., Chen Y., Garcia N., Whiteman N.B., McKay L.G., Storm N., Malsick L.E., Honko A.N., Lelis F.J., Habibi S., Jenni S., Cai Y., Rennick L.J., Duprex W.P., McCarthy K.R., Lavine C.L., Zuo T., Lin J., Zuiani A., Feldman J., MacDonald E.A., Hauser B.M., Griffths A., Seaman M.S., Schmidt A.G., Chen B., Neuberg D., Bajic G., Harrison S.C, & Wesemann D.R. (2021). Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike. Cell, 184(19), 4969-4980.e15.

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Comprehensive PBMC Immunophenotyping Protocol

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Peripheral blood mononuclear cells (PBMCs) were stained in four panels containing anti-CD3-PacificBlue (PacBlue) (antibodies from BD Biosciences unless otherwise indicated), anti-CD3-Alexa700, anti-CD25-PE-Cy7, anti-CD38-PE, anti-HLA-DR-PE-Cy7, anti-CCR5-APC, anti-CD123-PerCP-Cy5.5, anti-CD16-PacBlue, anti-CD80-FITC, anti-CD83-PE, anti-CD86-APC, anti-PD1-FITC, anti-PD-L1-PE, anti-HLA class I-APC, anti-CD69-APC-Cy7; anti-CD4-Qdot655, anti-CD8-PE-Cy5.5, anti-CD14-Qdot605 (Invitrogen); anti-CD45RA-ECD, anti-CD127-PE, anti-HLA-DR-ECD, anti-CD20-ECD (Beckman Coulter); anti-CD11c-Alexa700 (eBioscience); and anti-CD27-APC-Cy7 (BioLegend). A staining reagent for dead cells (Invitrogen Aqua Live/Dead Fixable Stain) was included. Cells were then washed and fixed in PBS containing 1% paraformaldehyde or permeabilized using a FOXP3 Fix/Perm kit (BioLegend) according to the manufacturer's instructions, intracellularly stained with anti-Ki67-Alexa 488 (BD Biosciences), anti-FOXP3-PacBlue, anti-T-Bet-BV711 (BioLegend), and anti-Eomes-eFluor 660 (eBioscience), fixed, and analyzed with a LSR II cytometer (Becton Dickinson) and FlowJo.

Méndez-Lagares G., Lu D., Chen C., Terrault N., Segal M.R., Khalili M., Monto A., Shen H., Manos M.M., Lanier L.L., Ryan J.C., McCune J.M, & Hartigan-O'Connor D. (2017). Memory T-cell proliferation before HCV therapy predicts antiviral immune responses and treatment success. Journal of immunology (Baltimore, Md. : 1950), 200(3), 1124-1132.

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Phenotypic and Functional Profiling of T Cells

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For suppression assays, cells were washed with 0.1% (w/v) sodium azide/phosphate-buffered saline (Mediatech Cellgro) on day 7 of in vitro stimulation and were stained with anti-CD4 PECy5 (BD Pharmingen), anti-CD8 Pacific Blue (Biolegend), and anti-CD25 APCCy7 (BD Pharmingen. For surface phenotyping of cells, bulk PBMCs and enriched CD8+ T cells were stained with anti-CD3 Alexa 700 (BD Pharmingen), anti-CD8 AmCyan (BD Biosciences), anti-CD27 APCCy7 (Biolegend), anti-CD28 APC (BD Pharmingen), CD45RO Pacific Blue (Biolegend), anti-CD62L PECy5 (BD Pharmingen), and anti-CD57 PE (Southern Biotech). For intracellular staining of cytokines, cells were initially activated with 1 μL of leukocyte activation cocktail (BD Pharmingen) for 5 hours. Cells were surface stained with anti-CD8 APC (BD Biosciences), anti-CD4 PECy5 (BD Pharmingen) and anti-CD25 APCCy7 (BD Pharmingen) and permeabilized as described previously. Intracellular staining was performed using anti-IFNγ PECy7 (BD Pharmingen), anti-IL-17A PE (Ebioscience), anti-Granzyme B Alexa 700 (BD Pharmingen) and anti-Perforin Pacific Blue (BD Pharmingen). All cells were resuspended in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfiled, PA) for FACS analysis. Flow cytometric data were acquired on a 4-Laser, 17-color LSRII using FACSDiva software (Becton Dickinson). CFSE was detected in the FITC channel on the LSR.

Cunnusamy K., Baughman E.J., Franco J., Ortega S.B., Sinha S., Chaudhary P., Greenberg B.M., Frohman E.M, & Karandikar N.J. (2014). Disease Exacerbation of Multiple Sclerosis is Characterized by Loss of Terminally Differentiated Autoregulatory CD8+ T cells. Clinical immunology (Orlando, Fla.), 152(0), 115-126.

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Apoptosis Analysis of T Cell Subsets

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Peripheral blood mononuclear cells were cultured in media (RPMI containing 10% fetal bovine serum, 1% l-glutamine, 1% penicillin-streptomycin, and 1% HEPES) overnight. The next day cells were harvested, washed with staining buffer (PBS containing 1% BSA and 0.1% azide), and assessed for apoptosis using BD Pharmingin's Annexin V PE Apoptosis Detection Kit I. Antibodies used to stain T cells included anti-CD3 (Pacific Blue), anti-CD4 (Alexa Fluor 700), anti-CD62L (APC), anti-CD45RO (FITC; BD Pharmingen), and anti-CD27 (APC-Cy7; BioLegend). Antibodies and Annexin V PE were added to each tube in a working volume of 100 µL of 1× Annexin V Buffer. Cells were incubated at room temperature for 15 minutes before an additional 200 µL Annexin V Buffer was added to each flow tube to bring the final volume to 300 µL. Cells were immediately examined on an LSRII flow cytometer.

Azzam S., Schlatzer D., Maxwell S., Li X., Bazdar D., Chen Y., Asaad R., Barnholtz-Sloan J., Chance M.R, & Sieg S.F. (2016). Proteome and Protein Network Analyses of Memory T Cells Find Altered Translation and Cell Stress Signaling in Treated Human Immunodeficiency Virus Patients Exhibiting Poor CD4 Recovery. Open Forum Infectious Diseases, 3(2), ofw037.

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