Igm apc

Manufactured by Southern Biotech

IgM APC is a fluorescently-labeled antibody that binds to the IgM immunoglobulin. It is used in flow cytometry applications to identify and quantify IgM-expressing cells.

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Lab products found in correlation

Cd27 bv605, by BioLegend (2 mentions) Cd19 pe cy7, by BioLegend (2 mentions) Easysep pan b cell magnetic enrichment kit, by STEMCELL (2 mentions) Cd38 bb515, by BD (2 mentions) Live dead bv510, by Thermo Fisher Scientific (2 mentions) Macsquanttyto cartridge sorting platform, by Miltenyi Biotec (2 mentions) Cd3 bv510, by BD (2 mentions) Igg1 pe, by BD (2 mentions) Imag system, by BD (2 mentions) Igg3 pe, by BD (2 mentions) Cd19 percp cy5, by BD (2 mentions) Igg3 pe, by Southern Biotech (2 mentions) Igg1 pe, by Southern Biotech (2 mentions) Igm apc, by BD (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) Biotinylated anti cd43 clone s7, by BD (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions) Cd27 pe cy7, by BD (1 mentions) Igd percp cy5, by BD (1 mentions) Cd10 pacific blue, by BioLegend (1 mentions)

5 protocols using igm apc

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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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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Investigating B Cell Differentiation in PrimPol-Deficient Mice

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Single cells suspensions were prepared from the spleens of 8-week old PrimPol^Δ/Δ and their wild-type littermates. After the lysis of red blood cells, naïve splenic B cells were enriched by CD43 depletion using biotinylated anti-CD43 (Clone S7, BD Biosciences), and the IMag^® system (BD Biosciences), as described by the manufacturer. Purified B cells were labelled for 10 min at 37°C with 5 μM carboxy fluorescein diacetate succinimidyl ester (CFSE, Molecular Probes) in IMDM medium containing 2% FCS. After washing, cells were cultured in IMDM, supplemented with 8% FCS, 50 μM 2-mercapthoethanol, penicillin/streptomycin at a density of 10⁵ cells/well in 24 well plates. CSR to IgG3 and IgG1 was induced by exposure to LPS (50 μg/ml Escherichia Coli LPS, 055:B5, Sigma) or LPS/rIL-4 (rIL4 20 ng/ml). Four days later, the proliferative capacity was determined by CFSE dilution; CSR frequencies were determined by staining with CD19-PercpCy5.5 (BD), IgM-APC, and IgG3-PE (LPS cultures) or IgG1-PE (LPS/rIL4 cultures). IgM-APC, IgG1-PE and IgG3-PE were purchased from Southern Biotech. Data were acquired by flow cytometry (Fortessa^®, BD) and analysed using FlowJo software (Version: 10.0.8r1).

Pilzecker B., Buoninfante O.A., Pritchard C., Blomberg O.S., Huijbers I.J., van den Berk P.C, & Jacobs H. (2016). PrimPol prevents APOBEC/AID family mediated DNA mutagenesis. Nucleic Acids Research, 44(10), 4734-4744.

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B Cell Immunophenotyping by Flow Cytometry

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PBMCs were isolated from heparinized peripheral blood (~15 ml) with Ficoll-Paque Plus (GE Healthcare), and stained with the following fluorochrome-labeled antibodies: CD19 PE-Cy5, CD10 Pacific Blue (BioLegend), IgD PerCP-Cy5.5, CD27 PE-Cy7, CD38 Alexa Fluor 700 (BD Pharmingen), and IgM APC (Southern Biotech). PBMCs were fixed (3% paraformaldehyde) and permeabilized (0.1% Tween-20) prior to staining with goat anti-human ARID3a antibody [21 (link)] and a rabbit anti-goat IgG FITC secondary (Invitrogen). Gating for individual B cell subsets was described previously [3 (link)] and used with the following B (CD20⁺) cell subset markers: transitional (IgD⁺CD27⁻CD10⁺), naïve (IgD⁺CD27⁻CD10⁺), MZ-like Memory (IgD⁺CD27⁺), Memory (IgD⁺CD27⁺), and double-negative (DN) (IgD⁻CD27⁻) B cells. Non-B cells were excluded using the following markers on the fluorochrome, APC: T cells (CD3), Monocytes, macrophages, and granulocytes (CD14), NK cells, neutrophils, macrophage and dendritic cells (CD16), and NK and NKT cells (CD56). Isotype controls (Caltag, BD Pharmingen, and eBioscience) were used for gating. Data (500,000 events per sample) were collected using an LSRII (BD Biogenics) and FACSDiva (BD Biosciences) software version 4.1 and were analyzed using FlowJo (Tree Star) software version 9.5.2.

Ward J.M., Ratliff M.L., Dozmorov M.G., Wiley G., Guthridge J.M., Gaffney P.M., James J.A, & Webb C.F. (2016). Human effector B lymphocytes express ARID3a and secrete interferon alpha. Journal of autoimmunity, 75, 130-140.

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Induction of Class-Switching in Naïve B Cells

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Single cells suspensions were prepared from the spleen of 8 to 10-week-old C9orf82^ko/ko mice and their wild-type littermates. Following erythrocytes lysis, naïve splenic B cells were enriched by the depletion of CD43 expressing cell using biotinylated anti-CD43 antibody (Clone S7, BD Biosciences), BD IMag Streptavidin Particles Plus and the IMag system (BD Biosciences), as described by the manufacturer. To measure their proliferative capacity, naïve B cells were labelled for 10 min at 37°C with 5 μM Carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes) in IMDM medium containing 2% FCS. After washing, cells were cultured in complete IMDM medium at a density of 10⁵ cells/well in 24 well plates. CSR to IgG3 and IgG1 was induced by exposure to LPS (50 μg/ml Escherichia Coli LPS, 055:B5, Sigma) or LPS+rIL-4 (rIL4 20 ng/ml). Four days later, the cells were harvested and stained with CD19-PercpCy5.5 (BD), IgM-APC, and IgG3-PE (LPS cultures) or IgG1-PE (LPS/rIL4 cultures) to determine CSR frequency along with CFSE dilution as an indicator of cell multiplication. IgM-APC, IgG1-PE and IgG3-PE were purchased from Southern Biotech. Data were acquired by flow cytometry (Fortessa, BD) and analyzed using FlowJo software (Version: 10.0.8r1).

Aslam M.A., Alemdehy M.F., Pritchard C.E., Song J.Y., Muhaimin F.I., Wijdeven R.H., Huijbers I.J., Neefjes J, & Jacobs H. (2019). Towards an understanding of C9orf82 protein/CAAP1 function. PLoS ONE, 14(1), e0210526.

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