Cd27 apc

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CD27-APC is a labeling reagent that binds to the CD27 cell surface receptor. It is used to identify and enumerate CD27-expressing cells in flow cytometry applications.

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Anti-CD27-APC (10 citations) Anti-CD27-APC-Cy7 (8 citations) CD27 APC-Cy7 (8 citations) Anti-CD27-APC (Clone L.G.3A10, (2 citations) Anti-CD27–APC (clone O323; (2 citations) APC/Fire750-CD27 (2 citations) CD27–APC (clone M-T271, (2 citations) CD27-APC/fire 700 (1 citations)

14 protocols using cd27 apc

Multiparametric Flow Cytometry Profiling of PBMCs

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PBMCs were counted and allowed to rest for 2 h in FACS buffer (PBS containing 10% FCS, 5 mM EDTA and 2 mM azide). For each staining 1×10⁶ PBMCs were used. The staining includes the following markers: CD3-PECy5.5 (Beckman Coulter, USA), CD4-PECy7 (Biolegend, USA), CD8-APCCy7 (BD Biosciences, USA), CD45RA-eFluor605 (eBiosciences, USA), CD57-Pacific Blue (Biolegend), CD28⁻PETexasRed (Beckman Coulter), CD27⁻APC (Biolegend). All staining performed included a Live/Dead marker (Invitrogen, USA) to exclude false positive staining. The CD4/CD8 ratio was measured in whole blood to match with previous studies.^{16 (link)} Briefly, 100 μl whole blood were stained with CD3-PECy5.5, CD4-PECy7 and CD8-APCCy7 for 20 min at 4 degrees followed by incubation with lysis buffer (eBiosciences) to eliminate red blood cells. Flow Cytometry data were analyzed using FlowJo (Treestar, USA), FACSDiva (BD Biosciences) and Kaluza (Beckman Coulter) (Supplementary Figure S1).

Ng T.P., Camous X., Nyunt M.S., Vasudev A., Tan C.T., Feng L., Fulop T., Yap K.B, & Larbi A. (2015). Markers of T-cell senescence and physical frailty: insights from Singapore Longitudinal Ageing Studies. NPJ Aging and Mechanisms of Disease, 1, 15005-.

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Purification of Memory B Cells

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Post-Ficoll mononuclear cells from healthy donors were enriched for B cells by magnetic separation with CD19 or CD20 microbeads (Miltenyi Biotech) and stained with CD20-PE-Cy7, CD27-APC (both from Biolegend), CD38-FITC (BD Pharmingen), CD27-APC and IgA-PE (Southern Biotech) prior to purification. Single CD20+CD38^dimCD27+IgA+ and CD20+CD38^dimCD27−IgA+ memory-B cells were sorted on a FACSAria flow cytometer (BD Biosciences) into 96-well PCR plates and immediately frozen on dry ice.

Berkowska M.A., Schickel J.N., Grosserichter-Wagener C., de Ridder D., Ng Y.S., van Dongen J.J., Meffre E, & van Zelm M.C. (2015). Circulating human CD27-IgA+ memory-B cells recognize bacteria with polyreactive immunoglobulins. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1417-1426.

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Comprehensive Immune Profiling by Flow Cytometry

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Cells were washed with PBS and stained first with LIVE/DEAD stain followed by combinations of cell surface markers, including: CD3 APC-Fire750, CD4 PE-CF594, CD8 PerCP-Cy5.5, CD45RA PE-Cy7, CD27 APC, PD-1 PE, CD39 BB515, CCR7 BV421 (all BioLegend), TIGIT-BV421, CD57-BB515 (BD Biosciences). For staining of transcription factors surface Foxp3 Transcription Factor Staining Buffer (Invitrogen) was used, according to manufacturer’s instructions, to fix and permeabilize cells before staining with T-bet-BV786 (BD Biosciences) and Eomes-PE-eFluor 610 (Invitrogen). Samples were fixed with 2% PFA, acquired on either a CyAn (Beckman Coulter) or LSR Fortessa (BD Biosciences) flow cytometer and analysed using FlowJo v9.9.6 software (FlowJo, Ashland, OR, USA).

Wallace Z., Kopycinski J., Yang H., McCully M.L., Eggeling C., Chojnacki J, & Dorrell L. (2022). Immune mobilising T cell receptors redirect polyclonal CD8+ T cells in chronic HIV infection to form immunological synapses. Scientific Reports, 12, 18366.

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SARS-CoV-2 Antibody Profiling with Flow Cytometry

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Recombinant SARS-CoV-2-S1 protein produced in HEK cells (Creative Diagnostics, DAGC091) was covalently labeled using CruzFluor647 (Santa Cruz Biotechnology, sc-362620) according to the manufacturer’s instructions.
Using fluorescence-activated cell sorting we sorted viable single cells from freshly isolated peripheral blood mononuclear cells (PBMCs as 7AAD⁻CD19⁺CD27⁺CD38⁺ antibody-secreting cells (ASCs) or SARS-CoV2-S1-enriched 7AAD⁻CD19⁺CD27⁺ memory B cells (MBCs) into 96-well PCR plates. Staining was performed on ice for 25 minutes in PBS with 2 % FCS using the following antibodies: 7-AAD 1:400 (Thermo Fisher Scientific), CD19-BV786 1:20 (clone SJ25C1, BD Biosciences, 563326), CD27-PE 1:5 (clone M-T271, BD Biosciences, 555441), CD38-FITC 1:5 (clone HIT2, BD Biosciences, 560982), and SARS-CoV-S1-CF647 at 1 μg/ml for patients CV07, CV38, CV23, CV24, CV 38, CV48, CV-X1, CV-X2 and CV01 (second time point, fig. S1). The first patients (CV01 (first time point), CV03, and CV05) were stained with a divergent set of antibodies: CD19-PE 1:50 (clone HIB19, BioLegend, 302207), CD38-PEcy7 1:50 (clone HIT2, BioLegend, 303505), CD27-APC 1:50 (clone O323, BioLegend, 302809) and DAPI as viability dye.

Kreye J., Reincke S.M., Kornau H.C., Sánchez-Sendin E., Max Corman V., Liu H., Yuan M., Wu N.C., Zhu X., Lee C.C., Trimpert J., Höltje M., Dietert K., Stöffler L., von Wardenburg N., van Hoof S., Homeyer M.A., Hoffmann J., Abdelgawad A., Gruber A.D., Bertzbach L.D., Vladimirova D., Li L.Y., Barthel P.C., Skriner K., Hocke A.C., Hippenstiel S., Witzenrath M., Suttorp N., Kurth F., Franke C., Endres M., Schmitz D., Jeworowski L.M., Richter A., Schmidt M.L., Schwarz T., Müller M.A., Drosten C., Wendisch D., Sander L.E., Osterrieder N., Wilson I.A, & Prüss H. (2020). A SARS-CoV-2 neutralizing antibody protects from lung pathology in a COVID-19 hamster model. bioRxiv.

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Multicolor Flow Cytometry of PBMC

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We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4⁺, CD8⁺, and CD19⁺. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 10⁶ events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo^® software.

Tawhari I., Hallak P., Bin S., Yamani F., Safar-Boueri M., Irshad A., Leventhal J., Ansari M.J., Cravedi P, & Gallon L. (2022). Early calcineurin-inhibitor to belatacept conversion in steroid-free kidney transplant recipients. Frontiers in Immunology, 13, 1096881.

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Sorting COVID-19 Antibody-Producing Cells

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Flow cytometry analysis was performed as previously described.^{52 (link)} PBMCs were isolated from blood of COVID-19 convalescent subjects using human lymphocyte separation medium. Cells were stained and mixed with fluorescent-labeled DNA-barcoded antigens and other fluorescent antibodies, and sorted by FACS with a MoFlo Astrios EQ Flow Cytometer (Beckman). The following antibodies were used to label the B cell subgroups: CD19-PB (BioLegend, Cat 302232), CD27-APC (BioLegend, Cat 302810), CD38-PE (BioLegend, Cat 303506). CD19⁺ CD27⁺ memory B cells and CD19⁺ CD27^high CD38^high plasma B cells were sorted for further scRNA-seq.

He B., Liu S., Wang Y., Xu M., Cai W., Liu J., Bai W., Ye S., Ma Y., Hu H., Meng H., Sun T., Li Y., Luo H., Shi M., Du X., Zhao W., Chen S., Yang J., Zhu H., Jie Y., Yang Y., Guo D., Wang Q., Liu Y., Yan H., Wang M, & Chen Y.Q. (2021). Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq. Signal Transduction and Targeted Therapy, 6, 195.

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Urine and Blood B Cell Analysis in AAV

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Urine and blood samples were collected from ten AAV patients with active disease. Urine samples were prepared as described previously (11 (link)). Briefly, urine was diluted 1:1 in PBS and centrifuged at 1,800 rpm. The sediment was resuspended in PBS and mononuclear cells (MNCs) were isolated using lymphoprep (Axis-Shield, Oslo, Norway). Next, MNCs were resuspended in wash buffer and stained with anti-human CD19-PerCP-Cy5.5, CD45-BV605, CD27-APC (BioLegend, San Diego, CA, USA), CD3-BUV395, and CD38-BB515 (BD Biosciences) for 15 min at room temperature in the dark. Isotype-matched non-specific antibodies were used as negative controls. In parallel, blood samples were labeled with the aforementioned monoclonal antibodies. Afterwards, cells were treated with 10x diluted FACS lysing solution for 10 min, washed twice in wash buffer and immediately analyzed. Stained urine and blood samples were acquired on the LSR-II and data was analyzed using Kaluza 1.5a software. Figure 3A shows a representative gating example of both blood and urine. Three patients were excluded because no renal involvement was diagnosed and accordingly no B cells were present in the urine.

von Borstel A., Land J., Abdulahad W.H., Rutgers A., Stegeman C.A., Diepstra A., Heeringa P, & Sanders J.S. (2019). CD27+CD38hi B Cell Frequency During Remission Predicts Relapsing Disease in Granulomatosis With Polyangiitis Patients. Frontiers in Immunology, 10, 2221.

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Lymphocyte Subset Immunophenotyping

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Immunophenotyping of lymphocyte subpopulations was performed with the following antibodies: CD3-PerCP (clone: HIT3a, BioLegend), CD4-FITC (clone: RPA-T4, BioLegend), CD8-BV510 (clone: RPA-T8, BioLegend), CD45RA-PE-Cy7 (clone: HI100, BioLegend), CD27-APC (clone: M-T271, BioLegend), TCR aβ-PE (clone: IP2b, BioLegend), TCR γδ-BV421 (clone: B1, BioLegend), CD19-APC (clone: HIB19, BioLegend), CD27-V450 (clone: M-T271, BioLegend), IgD-AF488 (clone: IA6-2, BioLegend), CD24-PE (clone: ML5, BioLegend), and CD38-PerC P (clone: HIT2, BioLegend). The samples were acquired on a FACSCanto II flow cytometer, and the data were analyzed by FlowJo. All reference values were obtained from our recent study on peripheral lymphocyte phenotyping (17 (link)).

Huang Y., Fang S., Zeng T., Chen J., Yang L., Sun G., Dai R., An Y., Tang X., Dou Y., Zhao X, & Zhou L. (2022). Clinical and immunological characteristics of five patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in China–expanding the atypical phenotypes. Frontiers in Immunology, 13, 972746.

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Isolation and Characterization of B Cells

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Heparinized blood samples were obtained from normal donors and patients enrolled in clinical research protocols approved by the Human Subjects Protection Committee of the Dana-Farber Cancer Institute (DFCI), at UCSD and the Mayo Clinic (CLL Research Consortium), and through the ICGC (42 (link)) after obtaining written informed consent. Treatment indication for all 21 patients in the discovery cohort was determined based on iwCLL criteria (12 (link)), (44 (link)). Peripheral blood mononuclear cells (PBMC) from normal donors and patients were isolated by Ficoll/Hypaque density gradient centrifugation. Mononuclear cells were used fresh or cryopreserved with 10% DMSO FBS and stored in vapor-phase liquid nitrogen until the time of analysis. CD19+ B cells from normal volunteers and CLL samples with WBC ≤50 × 10⁹/L were isolated by immunomagnetic selection (Miltenyi Biotec, Auburn CA) and stained with anti CD19 PE (BioLegend) prior to FACS sorting for live single cells in the presence of DAPI. MBL cells and naïve and memory B cells from age-matched healthy donors were isolated as follows: cryopreserved PBMCs were thawed and stained with anti CD19 PE, CD5 FITC, and CD27 APC (BioLegend). Cells were gated for naïve B cells (CD19+; CD27-; CD5), memory B cells (CD19+; CD27+; CD5-) or MBL (CD19+; CD5+) (Supplementary Figure 7a).

Kretzmer H., Biran A., Purroy N., Lemvigh C.K., Clement K., Gruber M., Gu H., Rassenti L., Mohammad A.W., Lesnick C., Slager S.L., Braggio E., Shanafelt T.D., Kay N.E., Fernandes S.M., Brown J.R., Wang L., Li S., Livak K.J., Neuberg D.S., Klages S., Timmermann B., Kipps T.J., Campo E., Gnirke A., Wu C.J, & Meissner A. (2020). Preneoplastic Alterations Define CLL DNA Methylome and Persist through Disease Progression and Therapy. Blood Cancer Discovery, 2(1), 54-69.

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Lymphocyte Immunophenotyping Protocol

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Immunophenotyping of lymphocyte subpopulations was performed with the following antibodies: CD3-PerCP (clone: HIT3a, BioLegend), CD4-FITC (clone: RPA-T4, BioLegend), CD8-BV510 (clone: RPA-T8, BioLegend), CD45RA-PE-Cy7 (clone: HI100, BioLegend), CD27-APC (clone: M-T271, BioLegend), TCR aβ-PE (clone: IP2b, BioLegend), TCR γδ-BV421 (clone: B1, BioLegend), CD19-APC (clone: HIB19, BioLegend), CD27-V450 (clone: M-T271, BioLegend), IgD-AF488 (clone: IA6-2, BioLegend), CD24-PE (clone: ML5, BioLegend), and CD38-PerCP (clone: HIT2, BioLegend). The samples were acquired on a FACSCanto II flow cytometer (BD Biosciences), and the data were analyzed using FlowJo. All reference values were obtained from our recent study on peripheral lymphocyte phenotyping.⁹ (link)

Yang L., Xue X., Zeng T., Chen X., Zhao Q., Tang X., Yang J., An Y, & Zhao X. (2020). Novel biallelic TRNT1 mutations lead to atypical SIFD and multiple immune defects. Genes & Diseases, 7(1), 128-137.

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