Igm percp cy5

Manufactured by BD

IgM-PerCP-Cy5.5 is a fluorescent-labeled antibody used in flow cytometry applications. It binds to human IgM and is conjugated with the PerCP-Cy5.5 fluorescent dye, which can be detected using the appropriate laser and filter set.

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Lab products found in correlation

Cd14 bv510, by BioLegend (2 mentions) Cd3 bv510, by BioLegend (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by BD (2 mentions) Facsaria iiiu, by BD (2 mentions) Cd56 bv510, by BioLegend (2 mentions) Cd27 alexa fluor 488, by BioLegend (2 mentions) Cd38 apc cy7, by BioLegend (2 mentions) Iga alexa fluor 647, by Jackson ImmunoResearch (2 mentions) Cd19 ecd, by Beckman Coulter (2 mentions) Igd pe cy7, by BD (2 mentions) Cd11c pe cy7, by BD (1 mentions) Cd69 pe cy7, by BD (1 mentions) Cd19 apc cy7, by BD (1 mentions) Facsverse, by BD (1 mentions) Cd4 v500, by BD (1 mentions) Cd23 pe cy7, by Thermo Fisher Scientific (1 mentions) Cd19 v450, by BD (1 mentions) Cd45.2 v500, by BD (1 mentions) Cd45.2 apc, by BD (1 mentions) Igd pe, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PerCP-Cy5.5 (120 citations) IgM PerCP-Cy5.5 (G20-127, (2 citations) IgM-Cy5.5-PerCP (2 citations) Mouse anti-human IgM PerCP-Cy5.5 (G20-127, (2 citations)

5 protocols using igm percp cy5

Comprehensive Immune Cell Profiling

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The following antibodies were used: CD4-V500, CD11c-PE-Cy7, CD19-V450, CD19-APC-Cy7, CD69-PE-Cy7, IgM-PerCP-Cy5.5, CD45.2-V500, CD45.2-APC (BD Biosciences), IgD-PE, CD45.1-FITC, CD11b-PerCP-Cy5.5, CD21-PB, CD23-PE-Cy7, AA4.1-APC (eBiosciences), and CD86-PB, CD45.1-PB (BioLegend). Following red blood cell lysis, Fc receptor blockade, and staining, cells were processed on a BD FACSVerse or LSR II flow cytometer and analyzed using FlowJo v9.6.4 (Treestar).

Mills R.E., Lam V.C., Tan A., Cresalia N., Oksenberg N., Zikherman J., Anderson M., Weiss A, & Hermiston M.L. (2015). Unbiased modifier screen reveals that signal strength determines the regulatory role murine TLR9 plays in autoantibody production. Journal of immunology (Baltimore, Md. : 1950), 194(8), 3675-3686.

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

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Blood samples were screened for SARS-CoV-2 RNA and for antibodies against HIV, HBV and HCV. Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-treated whole blood by density gradient centrifugation (Ficoll-Paque PREMIUM, Sigma-Aldrich). After separation, PBMC were stained with Live/Dead Fixable Aqua (Invitrogen; Thermo Scientific) in 100 μL final volume diluted 1:500 at room temperature RT. After 20 min incubation cells were washed with PBS and unspecific bindings were saturated with 50 μL of 20% normal rabbit serum (Life technologies) in PBS . Following 20 min incubation at 4°C cells were washed with PBS and stained with SARS-CoV-2 S-protein labeled with Strep-Tactin®XT DY-488 (iba-lifesciences cat# 2-1562-050) for 30 min at 4°C. After incubation the following staining mix was used CD19 V421 (BD cat# 562440), IgM PerCP-Cy5.5 (BD cat# 561285), CD27 PE (BD cat# 340425), IgD-A700 (BD cat# 561302), CD3 PE-Cy7 (BioLegend cat# 300420), CD14 PE-Cy7 (BioLegend cat# 301814), CD56 PE-Cy7 (BioLegend cat# 318318) and cells were incubated at 4°C for additional 30 min. Stained MBCs were single cell-sorted with a BD FACS Aria III (BD Biosciences) into 384-well plates containing 3T3-CD40L feeder cells and were incubated with IL-2 and IL-21 for 14 days as previously described (Huang et al., 2013 (link)).

Andreano E., Nicastri E., Paciello I., Pileri P., Manganaro N., Piccini G., Manenti A., Pantano E., Kabanova A., Troisi M., Vacca F., Cardamone D., De Santi C., Torres J.L., Ozorowski G., Benincasa L., Jang H., Di Genova C., Depau L., Brunetti J., Agrati C., Capobianchi M.R., Castilletti C., Emiliozzi A., Fabbiani M., Montagnani F., Bracci L., Sautto G., Ross T.M., Montomoli E., Temperton N., Ward A.B., Sala C., Ippolito G, & Rappuoli R. (2021). Extremely potent human monoclonal antibodies from COVID-19 convalescent patients. Cell, 184(7), 1821-1835.e16.

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Comprehensive B cell immunophenotyping

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B1 cells were detected via mAB IgM PerCP-Cy5.5 (clone R6-60.2; 1:100; BD Biosciences), CD19 APC-H7 (BD Biosciences), CD5-AlexaFlour647 (clone YTSS 121.5.2; 1:50; Serotec, Kidlington, UK) and CD11b (clone M1/70; 1:500; BD Biosciences), which was visualized by a secondary goat anti rat Ig polyclonal Ab (1:500; BD Biosciences) conjugated with PE. B2 cells were identified using IgM-PerCP-Cy5.5 and CD19 APC-H7. Furthermore, mAB CD21/35 APC (clone 7G6; 1:500; BD Biosciences), CD23 PE and IgD FITC (both kindly provided by R. Förster) were used for a more detail B cell characterisation. Around 1x10⁶ cells were incubated with mAB CD45.1/Ly.1 PE (clone A20; 1:100; Biolegend) or IgM^a FITC (clone DS-1; 1:500; BD Biosciences) to identify the injected cells. Subpopulations of B cells were identified by mAB B220 (clone RA3-6B2; 1:1000; BD Biosciences), IgM PerCP-Cy 5.5, IgD FITC, IgA (clone C10-3; 1:500; BD Biosciences), CD80 PerCy5.5 (clone 16-10A1; 1:1000; BD Biosciences) and CCR9 PE (clone eBIOCW-1.2; 1:100; eBioscience). IgA was visualized by a polyclonal secondary goat anti-rat Ig (1:500; BD Biosciences) conjugated with APC. Injected CD45.1/Ly.1 cells were analysed by counting more than 1000 positive cells/ organ. BrdU was detected as described previously [35 (link)]. Flow cytometric analyses were performed using the FACS Canto (BD Biosciences).

Weiberg D., Basic M., Smoczek M., Bode U., Bornemann M, & Buettner M. (2018). Participation of the spleen in the IgA immune response in the gut. PLoS ONE, 13(10), e0205247.

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Isolation of SARS-CoV-2 Memory B Cells

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Cryopreserved PBMCs from COVID-19 convalescent donors were thawed and stained with DAPI (BD564907), CD14-BV510 (BioLegend 301842), CD3-BV510 (BioLegend 317332), CD56-BV510 (BioLegend 318340), CD19-ECD (Beckman Coulter IM2708U), CD21-BV711 (563163), IgA-Alexa Fluor 647 (Jackson Immunoresearch 109-606-011), IgD-PE-Cy7 (BD 561314), IgM-PerCP-Cy5.5 (BD561285), CD27-Alexa Fluor 488 (BioLegend 393204) and CD38-APC-Cy7 (BioLegend 303534). Stained cells were then sorted using the BD FACSAria IIIu in a BSL3 facility. This procedure involved gating out all but live CD19⁺CD14^-CD3^-CD56^-IgM^-IgD^- cells, and then gating on IgA to yield purified populations of IgA-producing and IgG-producing memory B cells (MBCs).

Dacon C., Peng L., Lin T.H., Tucker C., Lee C.C., Cong Y., Wang L., Purser L., Cooper A.J., Williams J.K., Pyo C.W., Yuan M., Kosik I., Hu Z., Zhao M., Mohan D., Peterson M., Skinner J., Dixit S., Kollins E., Huzella L., Perry D., Byrum R., Lembirik S., Murphy M., Zhang Y., Yang E.S., Chen M., Leung K., Weinberg R.S., Pegu A., Geraghty D.E., Davidson E., Doranz B.J., Douagi I., Moir S., Yewdell J.W., Schmaljohn C., Crompton P.D., Mascola J.R., Holbrook M.R., Nemazee D., Wilson I.A, & Tan J. (2023). Rare, convergent antibodies targeting the stem helix broadly neutralize diverse betacoronaviruses. Cell Host & Microbe, 31(1), 97-111.e12.

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Phenotyping Memory B Cells by Flow Cytometry

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Cryopreserved PBMCs were thawed and stained with the following panel: DAPI (BD564907), CD14-BV510 (BioLegend, 301842), CD3-BV510 (BioLegend, 317332), CD56-BV510 (BioLegend, 318340), CD19-ECD (Beckman Coulter, IM2708U), CD21-BV711 (BD, 563163), IgA-Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-011), IgD-PE-Cy7 (BD, 561314), and IgM-PerCP-Cy5.5 (BD, 561285), CD27-Alexa Fluor 488 (BioLegend, 393204) and CD38-APC-Cy7 (BioLegend, 303534). The cells were sorted using the BD FACSAria IIIu in a BSL3 facility and gated on live CD19⁺CD14⁻CD3⁻CD56⁻IgM⁻IgD⁻IgA⁺/IgA⁻ (memory B cells).

Dacon C., Tucker C., Peng L., Lee C.C., Lin T.H., Yuan M., Cong Y., Wang L., Purser L., Williams J.K., Pyo C.W., Kosik I., Hu Z., Zhao M., Mohan D., Cooper A., Peterson M., Skinner J., Dixit S., Kollins E., Huzella L., Perry D., Byrum R., Lembirik S., Zhang Y., Yang E.S., Chen M., Leung K., Weinberg R.S., Pegu A., Geraghty D.E., Davidson E., Douagi I., Moir S., Yewdell J.W., Schmaljohn C., Crompton P.D., Holbrook M.R., Nemazee D., Mascola J.R., Wilson I.A, & Tan J. (2022). Broadly neutralizing antibodies target the coronavirus fusion peptide. bioRxiv.

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