Cd27 bv421

Manufactured by BD

Sourced in United States

CD27-BV421 is a fluorochrome-conjugated antibody that binds to the CD27 surface antigen. CD27 is a co-stimulatory receptor involved in the regulation of T-cell and B-cell activation and proliferation. The BV421 fluorochrome allows for detection using flow cytometry.

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6 protocols using cd27 bv421

Immunophenotyping of Cryopreserved Samples

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Cryopreserved samples were thawed, washed, and resuspended in PBS. Surface staining was performed according to standard protocols as published before [18 (link)]. Immunophenotyping was performed using fluorochrome-conjugated anti-human monoclonal antibodies (mAb) as follows: CD3-BV450 (UCHT1), CD3-BV510 (UCHT1), CD4-Alexa Fluor 700 (RPA-T4), CD8-APC-Cy7 (SK1), CD27-BV421 (M-T271), CD45RO-APC (UCHL1), CD197 (CCR-7)-PE-Cy7 (3D12), and CD69-FITC (L78) (BD Biosciences); CD158b-PE-Cy7 (DX27) and TCR Vγ9-FITC (B3) (BioLegend); TCR Vδ1-FITC (TS8.2) (Thermo Scientific); and TCR pan γδ-PE (REA591) and TIM3-APC (F38-2E2) (Miltenyi Biotec). FACS CANTO (BD Biosciences, San Jose, CA, USA) was used to acquire samples, and FlowJo V10 (TreeStar) was used to analyze the results. The gating strategy is shown in Figure 1(a).
Manual gating was used to characterize individual samples, and subsequently, data were downsampled and merged (concatenated) for further visualization using dimensionality reduction algorithm plugin, t-Distributed Stochastic Neighbor Embedding (tSNE).

Gaballa A., Arruda L.C., Rådestad E, & Uhlin M. (2019). CD8+γδ T Cells Are More Frequent in CMV Seropositive Bone Marrow Grafts and Display Phenotype of an Adaptive Immune Response. Stem Cells International, 2019, 6348060.

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Immunophenotyping Immortalized B Cells

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Mock- or MCM-treated immortalized B cells or TAB were stained with the following antibodies or matched isotypes and analyzed on a FACS Aria III (BD): CD19 BV711 (clone SJ25C1, 0.06 µg/100 µl, catalog number 563036), CD20 AF700 (clone 2H7, 0.5 µg/100 µl, 560631), CD24 PE-CF594 (clone ML5, 1 µg/100 µl, 562405), CD27 BV421 (clone M-T271, 0.25 µg/100 µl, 562513), CD38 APC (clone HIT2, 0.125 µg/100 µl, 555462), CD138 PE (clone MI15, 0.125 µg/100 µl, 552026), IgD PE-Cy7 (clone IA6-2, 0.125 µg/100 µl, 561314), IgG FITC (clone G18-145, 0.125 µg/100 µl, 555786), IgM BV605 (clone G20-127, 0.5 µg/100 µl, 562977) (all BD biosciences). Live/dead cell exclusion was performed by addition of 7-AAD (5 µg/ml, Calbiochem) prior to acquisition of the samples. Data were analyzed using FlowJo 10.4.2 (FlowJo LLC). The gating strategy is shown in Supplementary Fig. 10.

Griss J., Bauer W., Wagner C., Simon M., Chen M., Grabmeier-Pfistershammer K., Maurer-Granofszky M., Roka F., Penz T., Bock C., Zhang G., Herlyn M., Glatz K., Läubli H., Mertz K.D., Petzelbauer P., Wiesner T., Hartl M., Pickl W.F., Somasundaram R., Steinberger P, & Wagner S.N. (2019). B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma. Nature Communications, 10, 4186.

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Immune-Phenotypic Characterization of CAR-T Cells

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Immune-phenotypic characterization of CAR-T products was performed by Flow Cytometry, using the BD FACSymphony^TM Flow Cytometer. Antibodies used in the panels tested included: Viability stain dye (Invitrogen), CD3 BV570 (Biolegend), CD4 BUV563 (BD), CD8 BUV496 (BD), CD19CAR FITC (Acro Biosystem), CD45RO BUV395 (BD), CD27 BV421 (BD), CCR7 BUV805 (BD), 41BB BV650 (BD), TCF7 PE (BD) and Ki67 PE-Cy5 (ThermoFisher). Flow Cytometry data were analyzed by FlowJo v. 10.7. The gating strategy consisted of a set of steps starting from lymphocytes, based on SSC-A and FSC-A; single cells, based on FCS-H and FSC-A; and live cells, based on MFI for Viability dye. Consecutively specific gating was done in each population of interest e.g. CD4+ T cells, CD8+ T cells, etc. Lastly, populations of interest were concatenated and exported for unbiased clustering analysis, which was done using the Rphenograph package (https://github.com/jacoblevine/PhenoGraph). Projection of the density of cells expressing markers evaluated (above mentioned) was visualized using a non-linear dimensionality reduction technique, Uniform Manifold Approximation, and Projection (UMAP). All correlations were done using Spearman correlation. All statistical comparisons were done using the Mann–Whitney U test. Statistical analysis was performed using Graph Pad Prism version 8.

Maschan M., Caimi P.F., Reese-Koc J., Sanchez G.P., Sharma A.A., Molostova O., Shelikhova L., Pershin D., Stepanov A., Muzalevskii Y., Suzart V.G., Otegbeye F., Wald D., Xiong Y., Wu D., Knight A., Oparaocha I., Ferencz B., Roy A., Worden A., Kruger W., Kadan M., Schneider D., Orentas R., Sekaly R.P., de Lima M, & Dropulić B. (2021). Multiple site place-of-care manufactured anti-CD19 CAR-T cells induce high remission rates in B-cell malignancy patients. Nature Communications, 12, 7200.

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Comprehensive Immune Cell Profiling

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Peripheral blood mononuclear cells (PBMCs) were acquired and then were stained for surface markers for 20 min in PBS containing fluorescent antibodies. Fluorescently labeled antibodies included CD3-PerCP-Cy5.5, CD25-PE, CD45RA-FITC, CD8-PerCP-Cy5.5, CD19-PerCP-Cy5.5 and CD56-PE-Cy7 (Tianjin Three arrows, China); CD4-APC-H7, CD8-BV510, CD127-BV421, CCR7-AF647, CD28-PE-Cy7, CD3-APC-H7, CD4-PE-Cy7, CXCR3-Alexa488, CXCR6-BV510, CXCR5-Alexa647, CCR4- BV421, PD-1-PE, CD45-APC-H7, CD27-BV421, IgD-BB515, IgM-BV510, CD38-APC, CD24-PE, and CD21-PE-Cy7 (BD, United States). The instrument settings and gating strategies were adopted from previous works (Supplementary Figure S1) (Yang et al., 2020 (link); Zhu et al., 2020 (link)). All experiments, including cell separation and sample preparation, were performed according to standardized experimental manuals. Samples were analyzed using CytoFLEX flow cytometer (Beckman, United States). Results are expressed as the proportion of cells expressing particular markers. T cell subsets, including cytotoxic T (Tc) cells, helper T (Th) cells, T follicular helper (Tfh) cells, regulatory T (Treg) cells, B cells and NK subsets were identified (Supplementary Table S1).

Jiang Y., Yang M., Zhang Y., Huang Y., Wu J., Xie Y., Wei Q., Liao Z, & Gu J. (2021). Dynamics of Adaptive Immune Cell and NK Cell Subsets in Patients With Ankylosing Spondylitis After IL-17A Inhibition by Secukinumab. Frontiers in Pharmacology, 12, 738316.

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Phenotyping of B Cell Subsets in CVID Patients

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Peripheral blood was obtained from 23 CVID patients (according to ESID criteria) and 17 healthy donors at La Paz University Hospital, after informed consent was obtained. The study was approved by the Ethics Committee of the Hospital, and adhered to the principles of the Declaration of Helsinki. Clinical data were obtained from hospital records.
B cell phenotype was performed staining whole blood with CD19 Percp Cy5.5, CD27 FITC, CD38 FITC (BD Biosciences) IgM Alexa Fluor 647 (Jackson Inmuno Research), and IgD PE (Southern Biotech) antibodies and analyzed on a FACS Canto flow cytometer (BD Biosciences).
Peripheral blood mononuclear cells (PBMCs) were obtained after Ficoll gradient centrifugation. Naïve (CD19⁺IgD⁺CD27⁻), unswitched memory USm (CD19⁺IgD⁺CD27⁺) and switched memory Sm (CD19⁺IgD⁻CD27⁺) B cells were sorted from viable PBMCs after staining with CD19 FITC (4G7BD), Ig D PE (Southern Biotech), CD27 BV421 (M-T271, BD) antibodies in a FACS Aria (BD Biosciences). Purity was confirmed to be >95% for selected fractions. Sorted cells were pelleted and stored at −80°C. Further details of the sorting strategy are depicted in Supplementary Figure 1.

del Pino-Molina L., Rodríguez-Ubreva J., Torres Canizales J., Coronel-Díaz M., Kulis M., Martín-Subero J.I., van der Burg M., Ballestar E, & López-Granados E. (2019). Impaired CpG Demethylation in Common Variable Immunodeficiency Associates With B Cell Phenotype and Proliferation Rate. Frontiers in Immunology, 10, 878.

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Phenotyping of T cell subsets

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T cell phenotyping was performed on fresh peripheral blood mononuclear cells (PBMCs) that were isolated by density gradient. Cells were resuspended in Brilliant Staining Buffer (BD Biosciences, 563794) and then stained with Fixable Viability Stain-PE (BD Biosciences, New Jersey, US, 564996) to exclude non-viable cells, and the following uorescently-conjugated monoclonal antibodies: CD3-FITC (BD Biosciences, 555332), CD4-BUV395 (BD Biosciences, 564724), CD8-BV510 (BD Biosciences, 563256), CD45RA-APC-CyTM 7 (BD Biosciences, 560674), CD27-BV421 (BD Biosciences, 562513), CD28-PE-CyTM 7 (BD Biosciences, 560684) and CD57-APC (BD Biosciences 560845). After incubation and washing, cells were analyzed by ow cytometry using BD LSR FortessaTM X-20 ow cytometer and BD FACSDivaTM software (BD Biosciences). Data were analyzed with FlowJoTM v10 software (TreeStar).
After analysis (Supplemental le 1) CD4 + and CD8 + T cell populations were identi ed as part of different phenotypes: CD8 + and CD4 + naïve T cells (TN) (CD45RA + CD27 + CD28 + CD57-), CD8 + and CD4 + TCM (CD45RA-CD27 + CD28 + CD57-); CD8 + and CD4 + TTM (CD45RA-CD27 + CD28-CD57-); CD8 + TEM (CD45RA-CD27-CD28+/-CD57-), CD4 + TEM TH1 (CD45RA-CD27-CD28-CD57-), CD4 + TEM TH2 (CD45RA-CD27-CD28 + CD57-), and CD8 + and CD4 + TEMRA (CD45RA + CD27-CD28-CD57+), as previously described 17, 18, 19, 20, 21, 22, 23, 24, 25 (Supplemental le 2).

Martínez-Román P., Cortegano I., Millares D.V., Crespo‐Bermejo C., Rallón N., Benito J.M., Lara‐Aguilar V., Arca-Lafuente S., Rickenbach M., Domínguez L., Ryan P., Santos I.d.l., Coiras M., Palladino C., Gaspar M., Fernández‐Rodríguez A., & Briz V. (2022). T cell maturation and senescence after HCV cure in HIV-HCV coinfected patients: a prospective study.

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