Cd27 bv605

Manufactured by BioLegend

Sourced in United States

CD27 BV605 is a fluorochrome-conjugated antibody that recognizes the CD27 cell surface antigen. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on various immune cell types. The BV605 fluorochrome is used for flow cytometric analysis.

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Lab products found in correlation

Facsaria 2, by BD (3 mentions) Cd14 bv510, by BioLegend (3 mentions) Cd3 bv510, by BioLegend (3 mentions) Igd pe cy7, by BD (3 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions) Il 21, by Thermo Fisher Scientific (2 mentions) 7 aad, by BD (2 mentions) Cd19 pe lt19, by Miltenyi Biotec (2 mentions) Cd138 allophycocyanin b b4, by Miltenyi Biotec (2 mentions) Cd38 af700, by BioLegend (2 mentions) Cd20 efluor v450 2h7, by Thermo Fisher Scientific (2 mentions) Cd27 af647 lt27, by Bio-Rad (2 mentions) Cd19 percp cy5.5 sj225c1, by BD (2 mentions) Cd24 fitc ml5, by BD (2 mentions) Cd3viogreen bw264 56, by Miltenyi Biotec (2 mentions) Unconjugatedgoat anti irf4 m 17, by Santa Cruz Biotechnology (2 mentions) Donkey anti goat igg af488, by Thermo Fisher Scientific (2 mentions) Hybridomax hybridoma growth supplement, by Gentaur (2 mentions) Mem amino acids solution, by Merck Group (2 mentions) Ifn α, by Merck Group (2 mentions)

Spelling variants of this product

Anti-CD27-BV605 (6 citations) CD27 BV605 (O323), (2 citations)

11 protocols using cd27 bv605

Multiparametric Flow Cytometry of B Cells

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Antibodies used were CD19 PE (LT19; Miltenyi), CD138 allophycocyanin
(B-B4; Miltenyi Biotec), CD38 PE-Cy7 (HB7; BD Biosciences), CD38 AF700 (HIT2;
Biolegend), CD20 efluor V450 (2H7; eBioscience); CD27 AF647 (LT27; AbD Serotec),
CD27 FITC (M-T271), CD19 PerCP-Cy5.5 (SJ225C1; BD Biosciences), CD19 PE-Cy7
(SJ225C1; BD Biosciences), CD24 FITC(ML5; BD Biosciences), CD84 PE (CD84.1.21;
Biolegend), CD38 PerCP-Cy5.5 (HIT2; BD Biosciences), CD95 BV421 (DX2;
Biolegend), CD20 APC-H7(L27; BD Biosciences), CD27 BV605 (O323; Biolegend), CD3
VioGreen (BW264/56; Miltenyi), Ki67 FITC (B56; BD Biosciences), unconjugated
goat anti-IRF4 (M-17; Santa Cruz) and donkey anti-goat IgG AF488 (Polyclonal;
Invitrogen). Controls were isotype-matched mouse mAbs. Annexin V FITC was from
eBioscience and 7-AAD from BD Biosciences.
Reagents included human IL-2 (Roche); IL-6 (Peprotech) and IFN-α
(Sigma); IL-21 (PeproTech); goat anti-human F(ab′)₂ fragments
(anti-IgM and -IgG; Jackson Immunoresearch); HybridoMax hybridoma growth
supplement (Gentaur); Lipid Mixture 1, chemically defined (200X) and MEM Amino
Acids Solution (50X; Sigma).

Arumugakani G., Stephenson S.J., Newton D.J., Rawstron A., Emery P., Doody G.M., McGonagle D, & Tooze R.M. (2017). Early Emergence of CD19-Negative Human Antibody-Secreting Cells at the Plasmablast to Plasma Cell Transition. The Journal of Immunology Author Choice, 198(12), 4618-4628.

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Comprehensive Multicolor Flow Cytometry for Plasma Cell Analysis

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Abs used were CD19 PE (LT19; Miltenyi Biotec), CD138 allophycocyanin (B-B4; Miltenyi Biotec), CD38 PE-Cy7 (HB7; BD Biosciences), CD38 AF700 (HIT2; BioLegend), CD20 eFluor V450 (2H7; eBioscience); CD27 AF647 (LT27; AbD Serotec), CD27 FITC (M-T271), CD19 PerCP-Cy5.5 (SJ225C1; BD Biosciences), CD19 PE-Cy7 (SJ225C1; BD Biosciences), CD24 FITC (ML5; BD Biosciences), CD84 PE (CD84.1.21; BioLegend), CD38 PerCP-Cy5.5 (HIT2; BD Biosciences), CD95 BV421 (DX2; BioLegend), CD20 allophycocyanin-H7 (L27; BD Biosciences), CD27 BV605 (O323; BioLegend), CD3 VioGreen (BW264/56; Miltenyi Biotec), Ki67 FITC (B56; BD Biosciences), unconjugated goat anti-IRF4 (M-17; Santa Cruz Biotechnology), and donkey anti-goat IgG AF488 (polyclonal; Invitrogen). Controls were isotype-matched mouse mAbs. Annexin V FITC was from eBioscience, and 7-AAD was from BD Biosciences.
Reagents included human IL-2 (Roche), IL-6 (PeproTech), IFN-α (Sigma), IL-21 (PeproTech), goat anti-human F(ab′)₂ fragments (anti-IgM and anti-IgG; Jackson ImmunoResearch), HybridoMax hybridoma growth supplement (Gentaur), Lipid Mixture 1, Chemically Defined (200×), and MEM Amino Acids Solution (50×, Sigma).

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Antigen-Specific B Cell Sorting

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PBMCs were thawed and B cells were enriched using EasySep™ pan B cell magnetic enrichment kit (STEMCELL). B cells were stained with a panel containing CD19 PE-Cy7 (Biolegend), IgM APC (Southern Biotech), CD27 BV605 (Biolegend), CD38 BB515 (BD Biosciences), and CD3 BV510 (BD Biosciences). B cells were stained with surface stain master mix and each COVID-19 antigen probe for 30 minutes on ice in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin. Cells were stained with probe at a 1:100 dilution (NP, ORF8, RBD, PUUV, empty PE-SA) or 1:200 dilution (spike, endemic HCoV spikes). Cells were subsequently washed with 1X PBS 0.2% BSA and stained with Live/Dead BV510 (Thermo Fisher) in 1X PBS for 15 minutes. Cells were washed again and re-suspended at a maximum of 4 million cells/mL in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin for downstream cell sorting using the MACSQuantTyto cartridge sorting platform (Miltenyi). Cells that were viable/CD19⁺/antigen-PE⁺ or viable/CD19⁺/antigen-APC⁺ were sorted as probe positive. The PE⁺ and APC⁺ gates were drawn by use of FMO controls. Cells were then collected from the cartridge sorting chamber and used for downstream 10X Genomics analysis.

Dugan H.L., Stamper C.T., Li L., Changrob S., Asby N.W., Halfmann P.J., Zheng N.Y., Huang M., Shaw D.G., Cobb M.S., Erickson S.A., Guthmiller J.J., Stovicek O., Wang J., Winkler E.S., Madariaga M.L., Shanmugarajah K., Jansen M.O., Amanat F., Stewart I., Utset H.A., Huang J., Nelson C.A., Dai Y.N., Hall P.D., Jedrzejczak R.P., Joachimiak A., Krammer F., Diamond M.S., Fremont D.H., Kawaoka Y, & Wilson P.C. (2021). Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets. Immunity, 54(6), 1290-1303.e7.

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Multiparametric Flow Cytometry of Tonsil Cells

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The following titrated, conjugated antibodies were used for flow cytometry analysis: CD3-H7APC (SK7, BD Biosciences), CD4-BV650 (SK3, BD Biosciences), CD8-Pacific Blue (RPA-T8, BD Pharmingen), CD19-FITC (J3-119, Beckman-Coulter), CD27-BV605 (O323, Biolegend), CD45RO-Cy5PE (UCHL1, BD Pharmingen), PD-1-BV711 (EH12.2H7, Biolegend), CD57-AF594 (NK-1, Novus Bio), IgD-PE (Goat polyclonal, Southern Biotech), CD20-BV570 (2H7, Biolegend), CD38-BV786 (HIT2, Biolegend). 1-2×10⁶ tonsil-derived cells were thawed and rested for 2h in a cell culture incubator before staining. Cells were washed with PBS, BSA (0.5%), incubated (5 min, RT) with a viability dye (Aqua-dye, Invitrogen) and surface stained with titrated amounts of antibodies for 30 min at room temperature. After washing, cells were resuspended and fixed with 1% paraformaldehyde.

Oyler B.L., Valencia-Dávila J.A., Moysi E., Molyvdas A., Ioannidou K., March K., Ambrozak D., De Leval L., Fabozzi G., Woods A.S., Koup R.A, & Petrovas C. (2023). Multilevel human secondary lymphoid immune system compartmentalization revealed by complementary imaging approaches. iScience, 26(8), 107261.

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Immunophenotyping of Whole Blood Samples

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Venous blood was drawn into an ethylenediaminetetraacetic acid (EDTA) S-monovette (Sarstedt, Nümbrecht, Germany) for immunophenotyping. Whole blood (100 µl per flow cytometry panel) was directly stained in FACS tubes with fluorescence-labeled antibodies (BD-Biosciences, Franklin Lakes, United States: CD8-PerCP-Cy5.5, CD5-PE, CD45RA-PE-CF594, CD19-PE-Cy5, CD4-PE-Cy7, CD45RO-APC, CD38-A700, CD3-APC-H7, CD138-BV421, CD10-BV510, CD27-BV605, CD14-PE-CF594, CD19-PE-Cy5, CD25-PE-Cy7, CD16-A700, CD3-APC-H7, CD11b-BV421, HLADR-BV510; Biolegend, San Diego, United States: CD11c-BV605; Miltenyi Biotec, Bergisch-Gladbach, Germany: BDCA1-APC, BDCA2-PE) for 15 min in the dark at room temperature (RT). After washing, red blood cell (RBC) lysis was performed with 2 ml ACK lysing buffer (Thermo Scientific, Waltham, United States) for 7 min in the dark at RT. 2 ml of FACS buffer (phosphate buffered saline, 3% fetal calf serum, 1% sodium azide) was added to wash the samples twice. Subsequently, cells were resuspended in 400 µl FACS-buffer. After the staining procedure, cells were measured by flow cytometry (LSR Fortessa cytometer, BD Biosciences, Franklin Lakes, United States) and analyzed with FACS-Diva Software (BD Biosciences, Franklin Lakes, United States). The gating strategy is available in the supplementary data section of this paper (Supplementary Figure S7).

Shalchi-Amirkhiz P., Bensch T., Proschmann U., Stock A.K., Ziemssen T, & Akgün K. (2023). Pilot study on the influence of acute alcohol exposure on biophysical parameters of leukocytes. Frontiers in Molecular Biosciences, 10, 1243155.

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Immunophenotyping of Lymph Node-Derived Lymphocytes

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Lymph node-derived lymphocytes were stained with Aqua and the following titrated antibodies: CD3-Cy7APC, CD20-BV570, IgD-PE (Southern Biotech, cat no.2030-02), IgM-Cy5PE (clone G20-127, BD Biosciences), IgG-APC (clone G18-145, BD Biosciences), CD27-BV605 (clone O323, Biolegend), PNA-PE (GeneTex), CD86-FITC (IT2.2, BD Biosciences) and CXCR4-Cy7PE. In all, 2–3 × 10⁶ cells were stained with Aqua and biotinylated Env gp120 probe on ice for 30 min. Following a staining step with streptavidin, cells were surface-stained with antibodies and peanut agglutinin (PNA). The staining step was followed by washing and fixation using 1% paraformaldehyde (gating strategy in Supplementary Figure 5d).

Eslamizar L., Petrovas C., Leggat D.J., Furr K., Lifton M.L., Levine G., Ma S., Fletez-Brant C., Hoyland W., Prabhakaran M., Narpala S., Boswell K., Yamamoto T., Liao H.X., Pickup D., Ramsburg E., Sutherland L., McDermott A., Roederer M., Montefiori D., Koup R.A., Haynes B.F., Letvin N.L, & Santra S. (2021). Recombinant MVA-prime elicits neutralizing antibody responses by inducing antigen-specific B cells in the germinal center. NPJ Vaccines, 6, 15.

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Phenotyping PfCSP+ Memory B Cells

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Cryopreserved PBMCs from the VRC 314 trial were thawed and stained for 30 minutes at room temperature in the dark with the following panel: 1:500 Blue LIVE/DEAD (Thermo Fisher Scientific L23105), 18 μg/mL IgA-Dylight 405 (Jackson Immunoresearch 109-475-011), 1:50 CD14-BV510 (BioLegend 301842), 1:50 CD3-BV510 (BioLegend 317332), 1:100 CD8-BV510 (301048), 1:50 CD56-BV510 (BioLegend 318340), 1:50 CD27-BV605 (BioLegend 302830), 1:40 CD21-BV711 (BD Biosciences 563163), 1:50 CD19-BV750 (BioLegend 302236), 1:125 IgD-PECy7 (BD Biosciences 561314), 1:20 IgM-Brilliant Ultraviolet (BUV)395 (BD Biosciences 563903), 1:125 CD38-Alexa Fluor 700 (BD Biosciences 560676), 1:40 IgG-allophycocyanin (APC)H7 (BD Biosciences 561297), 1:25 PfCSP-Brilliant Blue (BB)660, and 1:25 PfCSP-BUV737. The PfCSP probes were made by conjugating biotinylated PfCSP to streptavidin tagged with the relevant fluorescent dye. The cells were sorted using a BD FACS Aria II and analyzed with FlowJo software (Tree Star). Cells were gated on live CD19⁺CD14^-CD3^-CD8^-CD56^-CD21⁺CD27⁺IgA⁺/IgG⁺, with CD27⁺⁺CD38⁺⁺ cells excluded.

Tan J., Cho H., Pholcharee T., Pereira L.S., Doumbo S., Doumtabe D., Flynn B.J., Schön A., Kanatani S., Aylor S.O., Oyen D., Vistein R., Wang L., Dillon M., Skinner J., Peterson M., Li S., Idris A.H., Molina-Cruz A., Zhao M., Olano L.R., Lee P.J., Roth A., Sinnis P., Barillas-Mury C., Kayentao K., Ongoiba A., Francica J.R., Traore B., Wilson I.A., Seder R.A, & Crompton P.D. (2021). Functional human IgA targets a conserved site on malaria sporozoites. Science translational medicine, 13(599), eabg2344.

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SARS-CoV-2 Spike and RBD Staining of PBMCs

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PBMCs were stained with SARS-CoV-2 Spike and RBD probes, as previously described (Juno et al., 2020 (link)). Probes were generated by sequential addition of streptavidin-phycoerythrin (PE) (Thermo Fisher Scientific) to trimeric S protein biotinylated using recombinant Bir-A (Avidity), while SARS-CoV-2 RBD was labeled to APC using an APC Conjugation Lightning-Link Kit (Abcam). PBMCs were surface stained with Aqua viability dye (Thermo Fisher) and monoclonal antibodies against CD19-ECD (#IM2708U, Beckman Coulter), IgM BUV395 (#563903), CD21 BUV737 (#564437), IgD PE-Cy7 (#561314), IgG BV786 (#564230), streptavidin-BV510 (#563261) (BD Biosciences), CD20 APC-Cy7 (#302314), CD14 BV510 (#301841), CD3 BV510 (#317332), CD8a BV510 (#301048), CD16 BV510 (#302048), CD10 BV510 (#312220) and CD27 BV605 (#302829) (BioLegend). Cells were washed, fixed with 1% formaldehyde and acquired on a BD LSRII Fortessa.

Nguyen T.H., Rowntree L.C., Petersen J., Chua B.Y., Hensen L., Kedzierski L., van de Sandt C.E., Chaurasia P., Tan H.X., Habel J.R., Zhang W., Allen L.F., Earnest L., Mak K.Y., Juno J.A., Wragg K., Mordant F.L., Amanat F., Krammer F., Mifsud N.A., Doolan D.L., Flanagan K.L., Sonda S., Kaur J., Wakim L.M., Westall G.P., James F., Mouhtouris E., Gordon C.L., Holmes N.E., Smibert O.C., Trubiano J.A., Cheng A.C., Harcourt P., Clifton P., Crawford J.C., Thomas P.G., Wheatley A.K., Kent S.J., Rossjohn J., Torresi J, & Kedzierska K. (2021). CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity. Immunity, 54(5), 1066-1082.e5.

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Enrichment and Sorting of SARS-CoV-2 Antigen-Specific B Cells

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PBMCs were thawed and B cells were enriched using EasySep™ pan B cell magnetic enrichment kit (STEMCELL). B cells were stained with a panel containing CD19 PE-Cy7 (Biolegend), IgM APC (Southern Biotech), CD27 BV605 (Biolegend), CD38 BB515 (BD Biosciences), and CD3 BV510 (BD Biosciences). B cells were stained with surface stain master mix and each COVID-19 antigen probe for 30 minutes on ice in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin. Cells were stained with probe at a 1:100 dilution (NP, ORF7a, ORF8, RBD) or 1:200 dilution (spike). Cells were subsequently washed with 1X PBS 0.2% BSA and stained with Live/Dead BV510 (Thermo Fisher) in 1X PBS for 15 minutes. Cells were washed again and re-suspended at a maximum of 4 million cells/mL in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin for downstream cell sorting using the MACSQuantTyto cartridge sorting platform (Miltenyi). Cells that were viable/CD19⁺/antigen-PE⁺ were sorted as probe positive. The PE⁺ gate was drawn by use of FMO controls. Cells were then collected from the cartridge sorting chamber and used for downstream 10X Genomics analysis.

Stamper C.T., Dugan H.L., Li L., Asby N.W., Halfmann P.J., Guthmiller J.J., Zheng N.Y., Huang M., Stovicek O., Wang J., Madariaga M.L., Shanmugarajah K., Jansen M.O., Amanat F., Stewart I., Changrob S., Utset H.A., Huang J., Nelson C.A., Dai Y.N., Hall P.D., Jedrzejczak R.P., Joachimiak A., Krammer F., Fremont D.H., Kawaoka Y, & Wilson P.C. (2020). Distinct B cell subsets give rise to antigen-specific antibody responses against SARS-CoV-2. Research Square.

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Phenotyping PfCSP+ Memory B Cells

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