Cd20 pe cy7

Manufactured by BioLegend

CD20-PE-Cy7 is a fluorochrome-conjugated monoclonal antibody that binds to the CD20 antigen expressed on B cells. It is a tool used in flow cytometry analysis for the identification and enumeration of B cells.

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CD20-PE (6 citations) CD20 PE-Cy7 (clone 2H7, (2 citations) PE-Cy7-conjugated anti-CD20 (2 citations) Anti-CD20 PE-Cy7 (2 citations)

5 protocols using cd20 pe cy7

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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Multicolor Immunofluorescence Analysis of FFPE Samples

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Formalin-fixed paraffin-embedded (FFPE) 4 µm slides were deparaffinized, rehydrated, and processed for heat-induced antigen retrieval. Samples were then washed with PBS and blocked against nonspecific binding using universal blocking buffer for 1 h at room temperature. Conjugated antibodies CD20/PE-cy7 (Clone 2H7, Biolegend, 1:50, Cat# 302312), BCL6/AF488 (Clone K112-91, BD Biosciences, 1:50, Cat# 561524) and SEMA4A/APC (Clone 5E3, Biolegend, 1:50, Cat# 148406) were diluted in 10% universal blocking buffer (5 µg/ml) and applied for 1 h at room temperature. Samples were then washed with PBS and mounted with antifade media and left to dry overnight at 4 °C. All images were acquired on Nikon A1 confocal microscope and analyzed using Nikon elements NIS.

Ruffin A.T., Cillo A.R., Tabib T., Liu A., Onkar S., Kunning S.R., Lampenfeld C., Atiya H.I., Abecassis I., Kürten C.H., Qi Z., Soose R., Duvvuri U., Kim S., Oesterrich S., Lafyatis R., Coffman L.G., Ferris R.L., Vignali D.A, & Bruno T.C. (2021). B cell signatures and tertiary lymphoid structures contribute to outcome in head and neck squamous cell carcinoma. Nature Communications, 12, 3349.

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Sorting of Naïve and Memory B Cells

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B cells were incubated with an antibody cocktail containing L/D marker (eBioscience Fixable Viability Dye eFluor 780) and antibodies CD10 BUV737 (BD #612826), CD20 PECy7 (BioLegend #302312) and CD27 BV711 (BioLegend #302834) and IgD BUV395 (BD #563813) for 30 min at 4°C. Labelled B cells were sorted into naïve (CD10⁻CD20⁺CD27⁻IgD⁺) and memory (CD10⁻CD20⁺CD27⁺ and CD10⁻CD20⁺CD27⁻IgD⁻) B cells using the FACSAria™ Fusion sorter (BD Biosciences) (gating strategy shown in Figure S1A).

Lee J.L., Fra‐Bido S.C., Burton A.R., Innocentin S., Hill D.L, & Linterman M.A. (2022). B cell‐intrinsic changes with age do not impact antibody‐secreting cell formation but delay B cell participation in the germinal centre reaction. Aging Cell, 21(9), e13692.

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Tetramer-based Identification and Sorting of B Cells

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B cells were eluted from PBMCs using a MACS Human B Cell isolation kit (Miltenyi Biotec). B cells were stained with rGP38 (IbAr10200) that had been tetramerized at 25 nM using Streptactin-PE (IBA Lifesciences) and Streptactin-APC (IBA Lifesciences). B cells were simultaneously stained with rGP38-Streptactin-PE and rGP38-Streptactin-APC tetramers for 1 hour on ice. Cells were washed twice in buffer (PBS, FBS, EDTA). Next, B cells were stained with a panel of antibodies. Donor 1 PBMCs were stained with a cocktail of anti-human CD3 PerCP-Cy5.5 (Biolegend), CD8 PerCP-Cy5.5 (Biolegend), CD14 PerCP-Cy5.5 (Invitrogen), CD16 PerCP-Cy5.5 (Biolegend), propidium iodide (PI) (Invitrogen), CD19 PE-Cy7 (Biolegend), CD27 BV510 (BD Biosciences), IgM BV711 (BD Biosciences), IgD BV421 (Biolegend), IgG BV605 (BD Biosciences), and IgA AF488 (Abcam). Donor 5 and 6 PBMCs were stained with a cocktail of anti-human CD3 PerCP-Cy5.5 (Biolegend), CD8 PerCP-Cy5.5 (Biolegend), CD14 PerCP-Cy5.5 (Invitrogen), CD16 PerCP-Cy5.5 (Biolegend), PI (Invitrogen), CD19 PE-Cy7 (Biolegend), CD20 PE-Cy7 (Biolegend), CD27 BV510 (BD Biosciences), IgM AF488 (Biolegend), and IgD BV421 (Biolegend). B cells were washed twice in buffer and run on a FACS Aria Fusion Cytometer (BD Biosciences). B cells were sorted into Super Script III reaction buffer (ThermoFisher Scientific) in 96-well Costar plates and frozen at −80 °C.

Shin O.S., Monticelli S.R., Hjorth C.K., Hornet V., Doyle M., Abelson D., Kuehne A.I., Wang A., Bakken R.R., Mishra A., Middlecamp M., Champney E., Stuart L., Maurer D.P., Li J., Berrigan J., Barajas J., Balinandi S., Lutwama J.J., Lobel L., Zeitlin L., Walker L.M., Dye J.M., Chandran K., Herbert A.S., Pauli N.T, & McLellan J.S. (2024). Crimean-Congo Hemorrhagic Fever Survivors Elicit Protective Non-Neutralizing Antibodies that Target 11 Overlapping Regions on Viral Glycoprotein GP38. bioRxiv.

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B-cell Expansion and TGFβ-1 Modulation

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Human B-cells were isolated from healthy donor PBMCs using the Easysep B-cell isolation kit (Stemcell Technologies). Isolated B-cells were cultured in ImmunoCult-XF T-cell Expansion Medium supplemented with ImmunoCult-ACF Human B-cell Expansion Supplement (Stemcell Technologies) for up to 7 days. For cells cultured with TGFβ−1, recombinant human TGFβ−1 (Peprotech, 10ng/mL) was added every day for the duration of the culture. For cells also cultured with SB431542 TGFβ receptor inhibitor, SB431542 (10uM) was 30 minutes before the addition of TGFβ−1.
Cells were labeled with Fixable Cell Proliferation Dye eFluor-450 (eBioscience) or stained with antibodies (all from Biolegend) CD19 PerCP-Cy5.5, CD22 Pacific Blue, CD72 APC, CD38 Alexa-Fluor700, and CD20 Pe-Cy7. In all experiments, cells were first incubated in Fc-Block (Biolegend), and dead cells and debris were excluded from analysis using Fixable Viability Dye eFluor780 (eBioscience). Cells were acquired by BD Symphony and analyzed using the FlowJo software.

Hou D., Castro B., Dapash M., Zolp A., Katz J., Arrieta V., Biermann J., Melms J., Kueckelhaus J., Benotmane J., Youngblood M., Rashidi A., Billingham L., Dmello C., Vazquez-Cervantes G., Lopez-Rosas A., Han Y., Patel R., Chia T.Y., Sun L., Prins R., Izar B., Heiland D.H., Zhang P., Sonabend A., Miska J., Lesniak M., Zhao J, & Lee-Chang C. (2023). B-cells Drive Response to PD-1 Blockade in Glioblastoma Upon Neutralization of TGFβ-mediated Immunosuppression. Research Square.

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