Anti cd20 fitc

Manufactured by BioLegend

Sourced in United States

Anti-CD20-FITC is a fluorochrome-conjugated antibody that binds to the CD20 surface antigen. CD20 is a pan-B cell marker expressed on B cells from the pre-B cell to the mature B cell stage of development. The FITC fluorochrome allows for the detection and analysis of CD20-positive cells by flow cytometry.

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Anti-CD20 (8 citations) CD20-FITC (5 citations) FITC anti-human CD20 (3 citations) FITC-conjugated fluorescently labelled anti-mouse CD20 (2 citations)

9 protocols using anti cd20 fitc

Characterization of B-cell Differentiation

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LN biopsy samples were minced and evaluated by two-color flow cytometry after staining in phosphate-buffered-saline without calcium chloride magnesium chloride (PBS (−)) with the following monoclonal antibodies: anti-CD19-phycoerythrin (PE) (BioLegend, San Diego, CA, USA), anti-CD27-PE (BioLegend), anti-CD3-PE (BioLegend), anti-CD20-fluorescein isothiocyanate (FITC) (BioLegend), and anti-CD38-FITC (BioLegend). BiPSCs were also evaluated by two-color flow cytometry before and after the induction of hematopoietic differentiation using the following monoclonal antibodies: anti-CD19-PE, anti-CD27-PE, anti-CD34-PE (BioLegend), anti-CD20-FITC, anti-CD38-FITC, anti-CD43-FITC (BioLegend), and anti-CD45-FITC (BioLegend). Immunofluorescence of the labeled cell membrane was evaluated using a BD FACSCANTO II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Kawamura F., Inaki M., Katafuchi A., Abe Y., Tsuyama N., Kurosu Y., Yanagi A., Higuchi M., Muto S., Yamaura T., Suzuki H., Noji H., Suzuki S., Yoshida M.A., Sasatani M., Kamiya K., Onodera M, & Sakai A. (2017). Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells. Scientific Reports, 7, 1659.

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Phenotyping Plasma Cells by Flow Cytometry

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Plasma cell cultures were labeled with 1:100 diluted anti–CD20-FITC (BioLegend, 302304), CD27-PE (BioLegend, 302808), CD138-APC (MI15, BioLegend, 356505), and CD38-APC.Cy7 (BioLegend, 303534) on ice for 30 minutes. A human myeloma cell line, U266 (ATCC), was used as a positive control for CD38 and CD138 expression. An isotype control (MOPC-21, BioLegend, 400121) was used to optimize the gating for CD138 (Figure 3B). CountBright Absolute Counting Beads (Life Technologies) were added for accurate cell count. Dead cell stain (7-AAD, BD Pharmingen, 559925) was added before acquisition to exclude dead cells from the analysis. All experiments were performed with the BD Biosciences FACSCanto flow cytometer and analyzed by FlowJo version 7.6.5 software (Tree Star).

Wong G.K., Barmettler S., Heather J.M., Millar D., Penny S.A., Huissoon A., Richter A, & Cobbold M. (2019). Aberrant X chromosome skewing and acquired clonal hematopoiesis in adult-onset common variable immunodeficiency. JCI Insight, 4(14), e127614.

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Multiparametric Flow Cytometry for Cell Purity

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Both primary cells and cell lines were tested using multiparametric flow cytometry for their purity.
Anti-CD10-PerCP/Cy5.5 (Cat no. 312215), anti-CD19-APC (Cat no.392503), anti-CD20-FITC (Cat no. 302303), and anti-CD38-PE (Cat no 356603) were purchased from Biolegend. Fluorescent viability dye, eFlour780, was purchased from ThermoFisher Scientific (Cat no. 65–0865-14). Cells were stained with individual antibodies (quantity as recommended by the manufacturer) in cold PBS containing 0.5% fetal calf serum and the fluorescent viability dye (1:1000 dilution) for 20 minutes. Cells were maintained at a density of one million cells per 100 ml of buffer. Cells were then washed three times with 200 μl of the buffer and resuspended at the above density. Control (unstained) cells were stained with just the fluorescent viability dye. Flow cytometry was performed to analyze the stained and control cells on a MACSQuant Analyzer, and the acquired data were further analyzed and plotted using FlowJo. At least 30,000 cells were acquired from each sample.

Paidi S.K., Raj P., Bordett R., Zhang C., Karandikar S.H., Pandey R, & Barman I. (2021). Raman and quantitative phase imaging allow morpho-molecular recognition of malignancy and stages of B-cell acute lymphoblastic leukemia. Biosensors & bioelectronics, 190, 113403.

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Evaluation of BAFF Signaling Pathway in B-cell

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CP-25 was supplied by the Chemistry Laboratory of Institute of Clinical Pharmacology of Anhui Medical University (Hefei, Anhui Province, China). Rituximab (Roche Pharma, Switzerland); Etanercept (Shanghai Guojian Pharmaceutical, Co., Ltd., China); BAFF (Pepro Tech, United States); anti-CD19-APC/PE/FITC, anti-CD20-FITC, anti-CD27-PE/APC, anti-BAFFR-APC, anti-BCMA-PE, and anti-TACI-PE (Biolegend, United States); anti-β-actin (ZSGB-BIO, China); Anti-TRAF2 (Santa Cruz, China); Anti-Phospho-NF-κBp65, anti-p100/52, and anti-Phospho-p38 MAPK (Cell Signaling Technology, United States); RPMI 1640 medium (Gibco, United States); fetal bovine serum (Zhejiang Tianhang Biotechnology, China).

Zhang F., Shu J.L., Li Y., Wu Y.J., Zhang X.Z., Han L., Tang X.Y., Wang C., Wang Q.T., Chen J.Y., Chang Y., Wu H.X., Zhang L.L, & Wei W. (2017). CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents. Frontiers in Pharmacology, 8, 933.

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Multiparametric Flow Cytometry of CD19+ B Cells

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Cultured CD19+ B cells were stained with a fixable viability dye (eFluor780, eBioscience, San Diego, CA, USA) in PBS for 30 min at 4 °C in the dark. For surface antigen staining, the cells were incubated with fluorochrome-conjugated antibodies (all from BioLegend, San Diego, CA, USA: anti-CD20-FITC, #302304; anti-CD20-PE, #302305; anti-CD39-BV421, #328214; anti-CD24-PE, #311106; anti-CD38-APC, #356606; or the respective isotype control) for 30 min at 4 °C in the dark. After washing and resuspending the cells in FACS Buffer (PBS + 2% FBS), the cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). To compensate for spillover effects, bead-based single stainings were performed. Dead cells and debris were excluded from the analysis based on viability dye fluorescence and scatter signals. The CD39high B-cell subset was defined as previously described [25 (link)]. Supplementary Figure S6 shows the gating strategy. All the collected data were evaluated using the FlowJo Software version 10.6.2 for Mac.

Coray M., Göldi V., Schmid L., Benecke L., Figueiró F, & Muller L. (2022). Differential Immunomodulatory Effects of Head and Neck Cancer-Derived Exosomes on B Cells in the Presence of ATP. International Journal of Molecular Sciences, 23(22), 14446.

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Isolation of CD19+ B cells from PBMCs

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Isolation methods were previously described by us or other colleagues [22 (link),25 (link),47 (link)]. Briefly, buffy coats of healthy individuals obtained from the Swiss Red Cross Blood Donation Center Basel were centrifuged on Ficoll–Paque Plus (GE Healthcare Biosciences, Chicago, IL, USA) to separate PBMCs by a density gradient. To remove adherent cells, the collected PBMCs were incubated for 30 min at 37 °C in RPMI 1640 medium (#11875093, Gibco, Life Technologies, Carlsbad, CA, USA) (supplemented with 1% penicillin/streptomycin and 10% FBS depleted of extracellular vesicles) before the magnetic separation. Using anti-CD19-antibody-coated magnetic beads (#130-050-301, Miltenyi Biotec, Bergisch Gladbach, Germany), CD19+ B cells were isolated from the nonadherent PBMCs by positive selection according to the manufacturer’s instructions. The purity of the B-cell isolation was determined by flow cytometry with anti-CD20 (anti-CD20 FITC (#302304) or anti-CD20-PE (#302305), BioLegend, San Diego, CA, USA). Purity values were generally > 90%.

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Immune Cell Profiling in Testes

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Immune cells in peripheral blood were collected and counts and phenotypes were analyzed by flow cytometry. The original data are reported in our previous studies (33 (link), 34 (link)). Interstitial cells in the testes were isolated as described previously (10 (link)). Briefly, testicular tissues were chopped and then incubated at 37°C with collagenase and DNase for 1 h. Tubule fragments were allowed to settle for 3 min, followed by interstitial cell recovery in PBS. Subsequently, the phenotypes of the interstitial cells were analyzed by multi-parameter flow cytometry. The anti-human flow cytometry antibodies cross-reactive with NPMs included anti-CD45-PE (557059, BD, USA), anti-CD3-APC-Cy7 (557757, BD), anti-CD20-FITC (302304, BioLegend, USA), anti-CD4-PerCP-Cy5.5 (317428, BioLegend), anti-CD8-PE-Cy7 (557746, BD), anti-Ki67-PE (51-36525X, BD), and anti-PD-1-PE (329906, BioLegend).

Song T.Z., Zhang M.X., Zhang H.D., Wang X.H., Pang W., Tian R.R, & Zheng Y.T. (2021). Glucose Metabolism Disorder Induces Spermatogenic Dysfunction in Northern Pig-Tailed Macaques (Macaca leonina) With Long-Term SIVmac239 Infection. Frontiers in Endocrinology, 12, 745984.

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Cryopreserved Xenograft Immune Profiling

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Cryopreserved xenografts were dissociated by mashing through a 30 μm cell strainer and blocked using 10% foetal bovine serum in PBS. Cells were resuspended in staining buffer consisting of 50% PBS containing 1% bovine serum albumin (BSA) and 50% Brilliant Stain Buffer (BD Biosciences) and incubated for 20 minutes at 4°C in the following antibodies: anti-CD45 (APC-H7; BD Biosciences, 560178), anti-CD3 (PE; BD Biosciences, 555333), anti-CD19 (PE-Cy7; Thermo Fisher Scientific, 25-0199-41), anti-CD20 (FITC; Biolegend, 302303), anti-IgK (BV421; Biolegend, 392705) and anti-IgL (APC; Biolegend, 316609). Cells were washed and re-suspended in flow cytometry buffer (PBS + 1% BSA) for flow cytometry. Cells were analysed using a BD LSRFortessa X-20 Cell Analyser (UCL Division of Medicine Core Facility, University College London) and data were analysed in FlowJo (v10).

Pearce D.R., Akarca A.U., De Maeyer R.P., Kostina E., Huebner A., Sivakumar M., Karasaki T., Shah K., Janes S.M., McGranahan N., Reddy V., Akbar A.N., Moore D.A., Marafioti T., Swanton C, & Hynds R.E. (2023). Phenotyping of lymphoproliferative tumours generated in xenografts of non-small cell lung cancer. Frontiers in Oncology, 13, 1156743.

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Detecting Apoptosis in IL-4 and Anti-CD40 Cultured Cells

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To validate some of the differentially expressed genes we fixed, permeabilized, and stained cells as previously described (7 (link)). The antibodies used were as follows; anti-IL4R APC (R&D), anti-CD27 FITC (Biolegend), anti-CD38 PE-CY7 (Biolegend), anti-CD20 FITC (Biolegend), anti-IRF4 alexa 647 (Invitrogen), anti-IRF8 APC (Biolegend), anti-BLIMP1 APC (R&D), and anti-active Caspase 3 alexa 647 (BD Biosciences). To determine the rates of apoptosis the IL-4 and anti-CD40 cultured cells were harvested and the dead cells removed using the Easysep dead cell removal kit (Stemcell). The cells were then recultured for 24 h with IL-4 and anti-CD40, followed by staining for Annexin V (eBioscience) and live/dead fixable violet dead stain kit (Life Technologies). Data was collected on a BD FACSCanto (BD Biosciences) and events were analyzed using FlowJo software version 10.4.2 (Tree Star).

Ramadani F., Bowen H., Gould H.J, & Fear D.J. (2019). Transcriptional Analysis of the Human IgE-Expressing Plasma Cell Differentiation Pathway. Frontiers in Immunology, 10, 402.

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