Percp cy5.5 anti cd27

Manufactured by BioLegend

PercP-Cy5.5-anti-CD27 is a fluorochrome-conjugated antibody that specifically binds to the CD27 molecule. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on T cells, B cells, and natural killer cells. The PercP-Cy5.5 fluorochrome is used for flow cytometric analysis.

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2 protocols using percp cy5.5 anti cd27

Sorting and Phenotyping of B Cell Subsets

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Peripheral B cells were purified from the blood of patients and control donors by positive selection using CD20 magnetic beads (Miltenyi Biotec). For sorting, enriched B cells were stained with FITC-anti-IgM, PE-anti-IgG, PE-Cy7-anti-CD10, APC-anti-CD21, Pacific Blue-anti-CD19, and PercP-Cy5.5-anti-CD27 (all from BioLegend) (gating strategy is shown in fig. S21). Single- or batch-sorted CD19⁺CD21^loCD10⁺IgM^hiCD27⁻ new emigrant and CD19⁺CD21⁺CD10⁻IgM⁺CD27⁻ mature naïve B cells from patients, heterozygous relatives, and HDs were sorted on a FACSAria flow cytometer (Becton Dickinson, Mountain View, CA) into 96-well polymerase chain reaction (PCR) plates or 5-ml round-bottom polystyrene test tubes, respectively. For phenotyping, enriched B cells were stained with FITC-anti-IgM, PE-anti-CD69, PE-Cy7-anti-CD10, APC-anti-CD86, PercP-Cy5.5-anti-CD27, APC-Cy7-anti-CD19 (all from BioLegend), and V450-anti-CD21 (BD Bioscience) (gating strategy is shown in fig. S22).

Sng J., Ayoglu B., Chen J.W., Schickel J.N., Ferre E.M., Glauzy S., Romberg N., Hoenig M., Cunningham-Rundles C., Utz P.J., Lionakis M.S, & Meffre E. (2019). AIRE expression controls the peripheral selection of autoreactive B cells. Science immunology, 4(34), eaav6778.

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Flow Cytometric Profiling of B Cell Subsets

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The following antibodies were used: FITC-anti-CD38 (BD Pharmingen), PE-anti-IgD (Southern Biotechnology Associates, Inc.), PE-anti-CD138 (Coulter-Immunotech), PerCP-Cy5.5-anti-CD27 (Biolegend), PE-Cy7-anti-CD21 (clone B-ly4, BD Pharmingen), PE-Cy7-anti-CD3 (Beckman Coulter), Cy5-anti-IgM (Jackson ImmunoResearch Laboratories, Inc.) and APC-H7-anti-CD19 (clone SJ25C1, BD Biosciences).
FACS CantoII and LSR II (BD Biosciences) cytometers were used to perform flow cytometric analysis. FACS data were analyzed using FlowJo (Tree Star Inc.) software.
For cell sorting PBMCs were sorted into CD19⁺IgM⁺CD21⁺CD38⁺CD27⁻ naïve B cells, CD19⁺IgM^hiCD21⁺ CD38⁺CD27⁺ MZ-like B cells, CD19⁺IgM⁻CD21⁺CD38⁺CD27⁺ switched memory B cells, CD19⁺IgM⁻CD21⁺CD38⁺CD27⁻ non-classical memory B cells and CD19⁺IgM^+/−CD21^low CD38^low B cells, as well as tonsillar B cells into CD19⁺IgD⁺CD27⁻CD38⁻ follicular naive B cells, CD19⁺IgD⁻CD27^−/+CD38⁺ GC B cells, CD19⁺IgD⁻CD27⁺⁺CD38⁺⁺CD138⁺ plasma cells (PC) and CD19⁺IgD⁻CD27⁺CD38⁻ memory B cell populations to a purity of >95% using a MoFlow cell sorter (Beckman Coulter).

Rakhmanov M., Sic H., Kienzler A.K., Fischer B., Rizzi M., Seidl M., Melkaoui K., Unger S., Moehle L., Schmit N.E., Deshmukh S.D., Ayata C.K., Schuh W., Zhang Z., Cosset F.L., Verhoeyen E., Peter H.H., Voll R.E., Salzer U., Eibel H, & Warnatz K. (2014). High Levels of SOX5 Decrease Proliferative Capacity of Human B Cells, but Permit Plasmablast Differentiation. PLoS ONE, 9(6), e100328.

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