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Anti cd20 percp cy5

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The Anti-CD20 PerCP-Cy5.5 is a fluorescent-conjugated antibody that binds to the CD20 antigen on the surface of B cells. It is designed for use in flow cytometry applications to identify and quantify B cell populations in biological samples.

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Lab products found in correlation

Anti cd56 pe, by Beckman Coulter (2 mentions) Anti pstat1 alexa488, by BD (2 mentions) Anti cd3 apc, by BD (2 mentions) Anti cd19 pe cy7, by BD (1 mentions) Anti cd38 fitc, by BD (1 mentions) Anti cd27 apc, by Thermo Fisher Scientific (1 mentions) Tri regent, by Merck Group (1 mentions) Facscanto 2, by BD (1 mentions) Facsaria cell sorter, by BD (1 mentions) Staining buffer, by BD (1 mentions) Facsdiva version 6, by BD (1 mentions) Lyse fix buffer, by BD (1 mentions) Perm buffer, by BD (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Anti cd19 bv421, by BD (1 mentions) Anti cd27 apc, by BD (1 mentions) Anti cd21 pe, by BD (1 mentions) Apc mouse igg1 k isotype control, by BD (1 mentions) Facsdiva software, by BD (1 mentions) Anti cd45 v500, by BD (1 mentions)

6 protocols using anti cd20 percp cy5

1

PBMC Immunophenotyping and Plasmablast Sorting

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After rapid thawing and washing, PBMCs were suspended in FACS buffer (PBS with 1% BSA). Specific mAbs or corresponding isotype fluorescence minus one (FMO) were added and kept at 4°C in the dark for 30 minutes, followed by washing twice with FACS buffer. Antibodies for FACS staining include anti-CD19 PE-Cy7, anti-CD20 PerCP-Cy5.5, and anti-CD38 FITC (BD Pharmingen) and anti-CD27 APC (eBioscience) [13 (link)]. Flow cytometry was performed on a FACSCanto II (Becton Dickenson) and data were analyzed using FlowJo software (TreeStar, Inc.). For plasmablast sorting, stained samples were sorted using a BD FACSAria cell sorter equipped with a 70μm nozzle into tubes containing 1ml of Tri Regent (Sigma) for RNA extraction.
Li L., Honda-Okubo Y., Li C., Sajkov D, & Petrovsky N. (2015). Delta Inulin Adjuvant Enhances Plasmablast Generation, Expression of Activation-Induced Cytidine Deaminase and B-Cell Affinity Maturation in Human Subjects Receiving Seasonal Influenza Vaccine. PLoS ONE, 10(7), e0132003.
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2

Flow Cytometry Analysis of pSTAT Signaling

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Fresh pre-warmed heparinized whole blood was stimulated in vitro with or without 600 ng/mL consensus sequence IFN-α (IFN-α-con1; InterMune, Brisbane, CA) for 5 min at 37°C. A 20-fold excess of Lyse/Fix buffer (BD Biosciences, San Jose, CA) was added for 10 min at 37°C for lymphocyte fixation and erythrocyte lysis. After centrifugation, cells were permeabilized with Perm Buffer (BD Biosciences) for 20 min on ice, washed twice, and resuspended in Staining Buffer (BD Biosciences).
All samples were stained with anti-CD56-PE (Beckman Coulter, Brea, CA) and anti-CD20-PerCP/Cy5.5 (BD Biosciences) to identify NK cells and B cells, respectively, and with anti-CD3-APC (both from BD Biosciences) to exclude T cells. Cells were additionally stained with anti-pSTAT1-Alexa488, or anti-pSTAT4-Alexa488 (BD Biosciences) for 20 min at room temperature. Samples were analyzed on an LSRII with FacsDiva Version 6.1.3 (BD Biosciences) and FlowJo Version 8.8.2 (Tree Star, Ashland, OR) software.
Werner J.M., Serti E., Chepa-Lotrea X., Stoltzfus J., Ahlenstiel G., Noureddin M., Feld J.J., Liang T.J., Rotman Y, & Rehermann B. (2014). Ribavirin Improves the IFN-γ Response of Natural Killer Cells to IFN-based Therapy of Hepatitis C Virus Infection. Hepatology (Baltimore, Md.), 60(4), 1160-1169.
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3

Phenotypic Characterization of B-Cell Subsets

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A total of 0.5 x 106 PBMCs were stained with the following combination of antibodies: Anti-CD19-BV421 (clone HIB19), anti-CD20-PerCPCy™5.5 (clone L27), anti-CD21-PE (clone B-ly4), anti-CD27-APC (clone M-T271) and anti-CD45-V500 (clone HI30). Anti-CD19-BV421, anti-CD20-PerCPCy™5.5, anti-CD21-PE, anti-CD27-APC, anti-CD45-V500, PerCP-Cy5.5 mouse IgG1 k isotype control, BV421 mouse IgG1 k isotype control, V500 mouse IgG1 k isotype control, PE mouse IgG1 k isotype control, and APC mouse IgG1 k isotype control were purchased from BD Biosciences (Becton Dickinson, Franklin Lakes, NJ, USA). B-cell populations were identified based on the expression of the following markers: activated memory B-cells (CD19+ CD20+ CD21- CD27+ CD45+), resting memory B-cells (CD19+ CD20+ CD21+ CD27+ CD45+), tissue-like memory cells (CD19+, CD20+ CD21- CD27- CD45+), and resting naïve B-cells (CD19+ CD20+ CD21+ CD27- CD45+).
CD34+ hematopoietic progenitors in peripheral blood were enumerated using the Stem Cell Enumeration Kit (Becton Dickinson).
All flow cytometry analyses were performed using a BD LSRFortessa™ Flow Cytometer (Becton Dickinson) from the I2MC cytometry platform (CHU Rangueil, Toulouse, France) and BD FACSDiva™ Software (Becton Dickinson).
Bonnefoy J., Baselet B., Moser D., Ghislin S., Miranda S., Riant E., Vermeesen R., Keiler A.M., Baatout S., Choukér A, & Frippiat J.P. (2022). B-Cell Homeostasis Is Maintained During Two Months of Head-Down Tilt Bed Rest With or Without Antioxidant Supplementation. Frontiers in Immunology, 13, 830662.
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4

Characterizing NK Cell Phenotype

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The following monoclonal antibodies were used to stain NK cells: anti-CD56-FITC, anti-CD3-PE, anti-CD20-PerCP-Cy5.5, and anti-CD14-APC (BD Biosciences). NK cells were stained with the antibodies for 30 min at 4 °C. Sample data were acquired on a FACS flow cytometer (BD FACSDiva™) and analyzed using BD FACSuite v1.2 software.
Park S.J., Yoon H.J., Gu E.Y., Lee B.S., Kim Y., Jung J., Kim J, & Moon K.S. (2022). A general toxicity and biodistribution study of human natural killer cells by single or repeated intravenous dose in severe combined immune deficient mice. Toxicological Research, 38(4), 545-555.
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5

Flow Cytometric Analysis of B Cells

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PBMCs were quickly thawed in pre-warmed, heat inactivated FBS, re-suspended in ice-cold MACS separation buffer (Milteyni Biotec, Auburn, California), counted, and enriched for B cells using the B Cell Isolation Kit II (Milteyni Biotec). Enriched B cells were then washed and re-suspended in PBS, divided in half, and stained with Live/Dead Violet dead cell dye (Life Technologies) for 30 minutes [59] . Each of the two samples were then washed and re-suspended in 2% FBS-PBS solution and stained with either AF488-HPV 16 psV or AF488-BPV psV, as well as the following fluorescent mAbs for 30 minutes: anti-CD38 APC and anti-IgD PE (Milteyni Biotec), anti-CD3 V500, anti-CD19 APC-Cy7, anti-CD27 PE-Cy7, and anti-CD20 PerCP-Cy5.5 (BD Biosciences, San Jose, California). All staining was conducted with optimized amounts of staining reagents, cells on ice, and minimal light exposure. Stained cells were washed and re-suspended in 2% FBS-PBS, maintained on ice, and protected from light until fluorescence activated cell sorting (FACS).
Scherer E.M., Smith R.A., Simonich C.A., Niyonzima N., Carter J.J, & Galloway D.A. (2014). Characteristics of Memory B Cells Elicited by a Highly Efficacious HPV Vaccine in Subjects with No Pre-existing Immunity. PLoS Pathogens, 10(10), e1004461.
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6

PBMC Immunophenotyping and Signaling Analysis

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Thawed PBMCs (1.5×106) were rested in 0.5 ml RPMI 1640 with 10% FBS, 1% penicillin/streptomycin and 2 mM L-glutamine (Mediatech) for 2h. Thereafter, cells were fixed with BD Cytofix buffer for 10 min at 37°C and 5% CO2, centrifuged, permeabilized with BD Perm buffer III for 20 min on ice, washed twice and resuspended in BD Phosflow Buffer (all from BD Biosciences). Cells were subsequently stained with anti-CD56-PE (Beckman Coulter, Brea, CA), anti-CD3-APC, anti-CD20-PerCP/Cy5.5 and anti-pSTAT1-Alexa488 (all from BD Biosciences) for 20 min at room temperature.
Alao H., Cam M., Keembiyehetty C., Zhang F., Serti E., Suarez D., Park H., Fourie N.H., Wright E.C., Henderson W.A., Li Q., Liang T.J., Rehermann B, & Ghany M.G. (2018). Baseline Intrahepatic and Peripheral Innate Immunity are Associated with Hepatitis C Virus Clearance During DAA Therapy. Hepatology (Baltimore, Md.), 68(6), 2078-2088.
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