Anti cd20 pecy7

Manufactured by BD

Anti-CD20-PECy7 is a fluorochrome-conjugated monoclonal antibody that binds to the CD20 antigen. It is designed for use in flow cytometry applications.

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Anti-CD20 (10 citations)

18 protocols using anti cd20 pecy7

Multiparametric Flow Cytometry Analysis

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The fluorochrome labeled monoclonal antibodies (mAbs) anti-CD2-FITC, anti-CD3-Alexa 700, anti-CD3-PerCP, anti-CD4-V450, anti-CD4-PE, anti-CD8-APC Fluor 780, anti-CD8-PacBlue, anti-CD16-FITC, anti-CD20 PECy7, anti-CD19-V450, anti-CD20-APCCy7, anti-CD25-PEcy7, anti- CD28-PE, anti-CD28-FITC, anti-CD38-PE, anti-CD39-FITC, anti-CD45-PerCP, anti-CD56-APC, anti-CD57-FITC, anti-CD197-PECy7, anti-HLA-DR, anti-IgM, anti-IgD, anti-Ki67-FITC, anti-Bcl-2-PE, anti-TNF-α-PE, anti-TNF-α-APC, IFN-γ-PcpCy5.5, and IL-2-PECy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-CD45RA-QDOT655 and anti-CD69-FITC mAb were obtained from Invitrogen (Carlsbad, CA). Anti-CD8-Alexa780, anti-CD27-Alexa Fluor 700, and anti-CD38-eFluor 650NC were obtained from E-bioscience (San Diego, CA). Anti-CD24-PEcy7 and anti-CD279-PE (PD-1) were purchased from Biolegend (San Diego, CA).
Human EBV protein and CMV peptide pool of pp65 sequence consisting of 138 peptides (15 mers with 11 amino acid overlaps) was purchased from JPT Peptide Technologies (Berlin, Germany).
All patients were DSA-free with a calculated panel reactive antibody (PRA) ≤20% at enrollment. Patient samples were assessed for donor-specific alloantibody post-transplantation as described previously ^{(18 (link))}.

Xu H., Samy K.P., Guasch A., Mead S.I., Ghali A., Mehta A., Stempora L, & Kirk A.D. (2015). Post Depletional Lymphocyte Reconstitution During Belatacept and Rapamycin Treatment in Kidney Transplant Recipients. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(2), 550-564.

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Isolation and Characterization of B Cells

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Frozen lymph node (LN) cell suspensions collected from macaques vaccinated with VLP-RC1–4fill^{12 (link)} were thawed and incubated in FACS buffer (1xPhosphate-buffered saline (PBS), 2% calf serum, 1 mM EDTA) with the following anti human antibodies at 1:200 dilution: anti CD3-APC-eFluor 780 (Invitrogen, 47-0037-41), anti CD14-APC-eFluor 780 (Invitrogen, 47-0149-42), anti CD16-APC-eFluor 780 (Invitrogen, 47-0168-41), anti CD8-APC-eFluor 780 (Invitrogen, 47-0086-42), anti CD20-PE-Cy7 (BD biosciences, 335793), and anti CD38 FITC (Stem Cell, 60131FI) and the LIVE/DEAD marker (Biolegend, 77184) at 1:400 dilution. Avi-tagged and biotinylated baits conjugated to fluorophore labeled streptavidin were added to the antibody mixture and incubated with the LN cells for 30 min. Single B cells were sorted into individual wells of a 96-well plates containing 5 μl of TCL lysis buffer (Qiagen, 1031576) and 1% beta-mercaptoethanol per well using a FACS Aria III (Becton Dickinson). The cell lysates were stored at −80°C or immediately used for subsequent mRNA purification.
To interrogate the B cell receptor specificity of the retrogenic B cells, the transduced B cells were incubated with Avi-tagged-biotinylated RC1 in complex with fluorophore labeled streptavidin for 30 min on ice. Alternatively, cells were incubated with fluorophore labeled streptavidin in the absence of RC1 for 30 min on ice.

Wang Z., Merkenschlager J., Chen S.T., Oliveira T.Y., Ramos V., Gordon K.M., Yao K.H., Jankovic M., Gazumyan A., Nussenzweig M, & Escolano A. (2019). Isolation of single HIV-1 Envelope specific B cells and antibody cloning from immunized rhesus macaques. Journal of immunological methods.

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Isolation and Characterization of SARS-CoV-2 Specific B Cells

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PBMCs were enriched for B cells by negative selection using a pan B cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were incubated in FACS buffer (1 X Phosphate-buffered Saline (PBS), 2% calf serum, 1 mM EDTA) with the following anti-human antibodies: anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41), anti-CD8-APC-eFluro 780 (Invitrogen, 47-0086-42), anti-CD16-APC-eFluro 780 (Invitrogen, 47-0168-41), anti-CD14-APC-eFluro 780 (Invitrogen, 47-0149-42), as well as Zombie NIR (BioLegend, 423105), and fluorophore-labeled RBD and Ovalbumin for 30 minutes on ice^{27 (link)}. Single CD3⁻CD8⁻CD16⁻CD20⁺Ova⁻RBD-PE⁺RBD-AF647⁺ B cells were sorted into individual wells of a 96-well plates containing 4 μl of lysis buffer (0.5 X PBS, 10mM DTT, 3000 units/mL RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III (Becton Dickinson). The sorted cells were frozen on dry ice, and then stored at −80°C or immediately used for subsequent RNA reverse transcription. Although cells were not stained for IgG expression, they are memory B cells based on the fact that they are CD20+ (a marker absent in plasmablasts) and they express IgG (since antibodies were amplified from these cells using IgG-specific primers).

Robbiani D.F., Gaebler C., Muecksch F., Lorenzi J.C., Wang Z., Cho A., Agudelo M., Barnes C.O., Gazumyan A., Finkin S., Hagglof T., Oliveira T.Y., Viant C., Hurley A., Hoffmann H.H., Millard K.G., Kost R.G., Cipolla M., Gordon K., Bianchini F., Chen S.T., Ramos V., Patel R., Dizon J., Shimeliovich I., Mendoza P., Hartweger H., Nogueira L., Pack M., Horowitz J., Schmidt F., Weisblum Y., Michailidis E., Ashbrook A.W., Waltari E., Pak J.E., Huey-Tubman K.E., Koranda N., Hoffman P.R., West AP J.r., Rice C.M., Hatziioannou T., Bjorkman P.J., Bieniasz P.D., Caskey M, & Nussenzweig M.C. (2020). Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals. bioRxiv.

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Isolation and Sorting of EDIII-Specific Memory B Cells

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PBMCs from sample 111 were enriched for B cells via positive selection using CD19 microbeads (Miltenyi Biotec; 130–050-301). PBMCs from all other donors were enriched for B cells by negative selection (Miltenyi Biotec; 130–101-638). All selection protocols were performed according to the manufacturer’s instructions. Enriched B cells were incubated for 30 min on ice in FACS buffer (1× PBS, 2% calf serum, 1 mM EDTA) with fluorophore-labeled EDIII and ovalbumin, and in the presence of anti-human antibodies anti-CD3-APC-eFluro 780 (Invitrogen; 47–0037-41), anti-CD8-APC-eFluro 780 (Invitrogen; 47–0086-42), anti-CD14-APC-eFluro 780 (Invitrogen; 47–0149-42), anti-CD16-APC-eFluro 780 (Invitrogen; 47–0168-41), anti-CD20-PECy7 (BD Biosciences; 335793), and Zombie NIR (BioLegend; 423105). Single CD3⁻CD8⁻CD14⁻CD16⁻ZombieNIR⁻CD20⁺Ova⁻EDIII-PE⁺EDIII-AF647⁺ B cells were sorted using a FACS Aria III (Becton Dickinson) into individual wells of 96-well plates. Each well contained 4 µl of a lysis buffer comprising 0.5× PBS, 10 mM DTT, and 3,000 U/ml RNasin Ribonuclease Inhibitors (Promega; N2615). Sorted cells were snap-frozen on dry ice and then stored at −80°C. Antibody sequences are derived from memory B cells because they originate from small CD20⁺ cells, and the antibody genes were PCR amplified using IgG-specific primers.

Agudelo M., Palus M., Keeffe J.R., Bianchini F., Svoboda P., Salát J., Peace A., Gazumyan A., Cipolla M., Kapoor T., Guidetti F., Yao K.H., Elsterová J., Teislerová D., Chrdle A., Hönig V., Oliveira T., West AP J.r., Lee Y.E., Rice C.M., MacDonald M.R., Bjorkman P.J., Růžek D., Robbiani D.F, & Nussenzweig M.C. (2021). Broad and potent neutralizing human antibodies to tick-borne flaviviruses protect mice from disease. The Journal of Experimental Medicine, 218(5), e20210236.

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Single-cell sorting for SARS-CoV-2 antibody discovery

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Single cell sorting by flow cytometry was performed as described (Robbiani et al., 2020 (link)). Briefly, peripheral blood mononuclear cells were enriched for B cells by negative selection using a pan-B-cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were incubated in FACS buffer (1× PBS, 2% FCS, 1 mM EDTA) with the following anti-human antibodies (all at 1:200 dilution): anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41), anti-CD8-APC-eFluor 780 (Invitrogen, 47-0086-42), anti-CD16-APC-eFluor 780 (Invitrogen, 47-0168-41), anti-CD14-APC-eFluor 780 (Invitrogen, 47-0149-42), as well as Zombie NIR (BioLegend, 423105) and fluorophore-labelled RBD and ovalbumin (Ova) for 30 min on ice. Single CD3−CD8−CD14−CD16−CD20+Ova−Gamma NTD-PE⁺Wuhan-Hu-1 NTD-AF647⁺ B cells were sorted into individual wells of 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/ml RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III and FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis. The sorted cells were frozen on dry ice, and then stored at −80 °C or immediately used for subsequent RNA reverse transcription.

Wang Z., Muecksch F., Cho A., Gaebler C., Hoffmann H.H., Ramos V., Zong S., Cipolla M., Johnson B., Schmidt F., DaSilva J., Bednarski E., Tanfous T.B., Raspe R., Yao K., Lee Y.E., Chen T., Turroja M., Milard K.G., Dizon J., Kaczynska A., Gazumyan A., Oliveira T.Y., Rice C.M., Caskey M., Bieniasz P.D., Hatziioannou T., Barnes C.O, & Nussenzweig M.C. (2022). Conserved Neutralizing Epitopes on the N-Terminal Domain of Variant SARS-CoV-2 Spike Proteins. bioRxiv.

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Characterizing iPSC-derived T Cells

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For detection of CD107a and IFNγ expression of iPSC-T cells, T cells were co-cultured with B-LCLs cells at a 1:1 ratio in a 96-well plate for 5 h in 100 μL of RPMI-1640 supplemented with glutamine, 1× Monensin (BioLegend), 10% FBS, and anti-CD107a-APC antibody (BioLegend, Clone H4A3, 1 μL). Then the cell surface was stained with anti-CD20-PE-Cy7 (BD Pharmingen) and anti-EGFR-PE (BioLegend). Samples were washed and fixed with Fixation Buffer (BioLegend) for 20 min. Samples were washed twice with Permeabilization Wash Buffer (BioLegend) and were stained with IFNγ-APC-Cy7 (BioLegend) for 20 min. The washed and resuspended cells were analyzed using BD FACSAria II.

Takayanagi S.I., Wang B., Hasegawa S., Nishikawa S., Fukumoto K., Nakano K., Chuganji S., Kato Y., Kamibayashi S., Minagawa A., Kunisato A., Nozawa H, & Kaneko S. (2023). Mini-TCRs: Truncated T cell receptors to generate T cells from induced pluripotent stem cells. Molecular Therapy. Methods & Clinical Development, 31, 101109.

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Isolation and Sorting of Memory B Cells for Sequencing

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PBMCs were separated from the whole-blood samples obtained from four volunteers using Histopaque (Sigma) gradient centrifugation. After washing with Hank’s balanced salt solution (HBSS) (Solarbio) for three times, the cells were aliquoted and stored in liquid nitrogen in the presence of FBS and DMSO. For single memory B cell sorting, stored PBMCs were thawed and incubated with CD19 MicroBeads (Miltenyi Biotec) to screen out CD19+ B lymphocytes, which were then incubated with human Fc block (BD Biosciences), anti-CD20-PECy7 (BD1113 Biosciences), S-ECD-PE, and S-ECD-APC. The single memory B cells (CD20-1114 PECy7+ S-ECD-PE+ S-ECD-APC+) were further sorted into 96-well plates using a FACSAria II (BD Biosciences), and followed by sequencing and cloning as previously described^{35 (link)}.

Wang K., Jia Z., Bao L., Wang L., Cao L., Chi H., Hu Y., Li Q., Zhou Y., Jiang Y., Zhu Q., Deng Y., Liu P., Wang N., Wang L., Liu M., Li Y., Zhu B., Fan K., Fu W., Yang P., Pei X., Cui Z., Qin L., Ge P., Wu J., Liu S., Chen Y., Huang W., Wang Q., Qin C.F., Wang Y., Qin C, & Wang X. (2022). Memory B cell repertoire from triple vaccinees against diverse SARS-CoV-2 variants. Nature, 603(7903), 919-925.

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Isolation and Characterization of SARS-CoV-2 RBD-Specific B Cells

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As previously described (Robbiani et al., 2020 (link)), PBMCs were enriched for B cells via negative selection using a pan–B cell isolation kit (130-101-638; Miltenyi Biotec) according to the manufacturer’s protocol. Enriched B cells were incubated with fluorophore-labeled RBD and ovalbumin, and in the presence of antihuman antibodies anti-CD3-APC-eFluro 780 (47-0037-41; Invitrogen), anti-CD8-APC-eFluro 780 (47-0086-42; Invitrogen), anti-CD14-APC-eFluro 780 (47-0149-42; Invitrogen), anti-CD16-APC-eFluro 780 (47-0168-41; Invitrogen), anti-CD20-PECy7 (335793; BD Biosciences), and Zombie NIR (423105; BioLegend) in FACS buffer (1 × PBS, 2% calf serum, 1mM EDTA) for 30 min on ice. Single CD3⁻CD8⁻CD14⁻CD16⁻ZombieNIR^-CD20⁺Ova⁻RBD⁺RBD KEN⁺ were sorted using a FACS Aria III (Becton Dickinson) into individual wells of a 96-well plate, each containing 4 μl of lysis buffer comprising 0.5× PBS, 10 mM dithiothreitol, and 3,000 U/ml RNasin Ribonuclease Inhibitors (N2615; Promega). Sorted cells were frozen on dry ice and stored at −80°C until further processing.

Agudelo M., Muecksch F., Schaefer-Babajew D., Cho A., DaSilva J., Bednarski E., Ramos V., Oliveira T.Y., Cipolla M., Gazumyan A., Zong S., Rodrigues D.A., Lira G.S., Conde L., Aguiar R.S., Ferreira OC J.r., Tanuri A., Affonso K.C., Galliez R.M., Castineiras T.M., Echevarria-Lima J., Bozza M.T., Vale A.M., Bieniasz P.D., Hatziioannou T, & Nussenzweig M.C. (2022). Plasma and memory antibody responses to Gamma SARS-CoV-2 provide limited cross-protection to other variants. The Journal of Experimental Medicine, 219(9), e20220367.

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Single-cell sorting of SARS-CoV-2 specific B cells

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Single cell sorting by flow cytometry was performed as described (Robbiani et al., 2020 (link)). Briefly, peripheral blood mononuclear cells were enriched for B cells by negative selection using a pan-B-cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were incubated in FACS buffer (1× PBS, 2% FCS, 1 mM EDTA) with the following anti-human antibodies (all at 1:200 dilution): anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41), anti-CD8-APC-eFluor 780 (Invitrogen, 47-0086-42), anti-CD16-APC-eFluor 780 (Invitrogen, 47-0168-41), anti-CD14-APC-eFluor 780 (Invitrogen, 47-0149-42), as well as Zombie NIR (BioLegend, 423105) and fluorophore-labelled RBD and ovalbumin (Ova) for 30 min on ice. Single CD3−CD8−CD14−CD16−CD20+Ova−Gamma NTD-PE⁺Wuhan-Hu-1 NTD-AF647⁺ B cells were sorted into individual wells of 96-well plates containing 4 μL of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/mL RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III and FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis. The sorted cells were frozen on dry ice, and then stored at −80 °C or immediately used for subsequent RNA reverse transcription.

Wang Z., Muecksch F., Cho A., Gaebler C., Hoffmann H.H., Ramos V., Zong S., Cipolla M., Johnson B., Schmidt F., DaSilva J., Bednarski E., Ben Tanfous T., Raspe R., Yao K., Lee Y.E., Chen T., Turroja M., Milard K.G., Dizon J., Kaczynska A., Gazumyan A., Oliveira T.Y., Rice C.M., Caskey M., Bieniasz P.D., Hatziioannou T., Barnes C.O, & Nussenzweig M.C. (2022). Analysis of memory B cells identifies conserved neutralizing epitopes on the N-terminal domain of variant SARS-Cov-2 spike proteins. Immunity, 55(6), 998-1012.e8.

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Multiparametric Flow Cytometry Protocol

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Monoclonal antibodies (mAbs) anti-CD3-Alexa-700, anti-CD3-PerCP, anti-CD4-V450, anti-CD4-PE, anti-CD8-PacBlue, anti-CD16-FITC, anti-CD20-PECy7, anti-CD20-APCCy7, anti-CD31-FITC, anti-CD45-PerCP, anti-CD56-APC, anti-CD57-FITC, anti-CD57-BV605, anti-CD127 (IL-7Rα)-APC, and anti-CCR7-PECy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-CD45RA-QDOT655 was obtained from Invitrogen (Carlsbad, CA). Anti-CD8-APC-eFluor-780 and anti-PD1-PE were purchased from BioLegend (San Diego, CA). Recombinant human IL-7 and IL-7 enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN).

Xu H., Bendersky V.A., Brennan T.V., Espinosa J.R, & Kirk A.D. (2017). IL-7 Receptor Heterogeneity as a Mechanism for Repertoire Change during Post-Depletional Homeostatic Proliferation and its Relation to Costimulation Blockade–Resistant Rejection. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 18(3), 720-730.

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