Igm fitc

Manufactured by Thermo Fisher Scientific

Sourced in United States

IgM-FITC is a fluorescent-labeled antibody that binds to the IgM class of immunoglobulins. It is commonly used in flow cytometry and immunofluorescence applications to detect and quantify the presence of IgM in biological samples.

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FITC-conjugated goat-anti-mouse IgM (4 citations) PE-anti-IgM (eB121-15F9); APC anti-CD93 (AA4.1); FITC-anti-F4/80 (BM8) (2 citations) Anti-IgM-FITC (9 citations) FITC-conjugated anti-IgM (3 citations) FITC-conjugated anti-murine IgM antibody (2 citations) Rat anti-mouse IgM FITC (2 citations) FITC-conjugated goat anti-human IgM or IgG antibody (2 citations)

7 protocols using igm fitc

Multicolor Flow Cytometry Panel

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Commercial antibodies (clones, fluorophores and sources) are as follows: CD3-FITC (145–2C11), CD62L-PE (Mel-14), CD4-PErCP/Cy5.5 (RM4–5), CD8-PerCP/Cy5.5 (53–6.7), CD8-APC (53–6.7), CD209b-APC (22D1), IgM-FITC (II/41), CD4-PE (GK1.5), CD25-AF488 (PC61.5), Streptavidin-PE, CD169-PE/Cy7 (3D6.112) (eBioscience, San Diego, CA); CD11b-FITC (M1/70), CD49d-FITC (R1–2), TCRβ-FITC (GL3), CD21/35-PE (7E9), CD23-APC (B3B4), Ly6G-PE (RB6–8C5), Ly6C-PerCP/Cy5.5 (HK1.4), CD11c-PE/Cy7 (N418), B220-PerCP/Cy5.5 (RA3–6B2), B220-APC (RA3–6B2), B220-APC/Cy7 (RA3–6B2), CD69-BV421 (H1.2F3),CD23-Biotin (B3B4), F4/80-APC (BM8), Ly6G-BV421 (1A8), CD45-Pacific Blue (30-F11), CD45-BV510 (30-F11), CD43-PE (1B11), CD24-PE/Cy7 (M1/69), IgD-Pacific Blue (11–26c.2a), CD69-PE/Cy7 (H1.2F3), IgD-PerCP/Cy5.5 (11–26c.2a), Ly51-AF647 (6C3), CD3-Pacific Blue (17A2), CD3-PE (17A2), TCRγ/δ-biotin (GL3), Streptavidin-PE/Cy7 (Biolegend, San Diego, CA); Siglec-F-PE (E50–2440) (BD Biosciences, San Jose, CA). Samples were preincubated with 1 μg Fc-block (2.4G2 hybridoma; ATCC).
Cells were acquired either on the BD Biosciences LSR Fortessa or with a BD FACScan flow cytometer with DxP multi-color upgrades by Cytek Development Inc. (Woodland Park, NJ) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR).

Anaya E.P., Lin X., Todd E.M., Szasz T.P, & Morley S.C. (2021). Novel mouse model reveals that serine phosphorylation of L-plastin is essential for effective splenic clearance of pneumococcus. Journal of immunology (Baltimore, Md. : 1950), 206(9), 2135-2145.

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Multicolor FACS Analysis of B Cell Subsets

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Cells were resuspended at a concentration of 10⁶ cells/100 μl fluorescence-activated cell sorting (FACS) buffer (1X PBS, 2 mM EDTA, 1% BSA). Cells were stained with Fc block (BD Biosciences) for fifteen minutes, stained with antibody-fluorophores for one hour on ice, then washed with ten volumes of FACS buffer. The following antibodies were used: B220-PE-Cy7 (RA3–6B2, Tonbo), CD11b-APC-Cy7 (M1/70, Tonbo), CD45.1-FITC (A20, Tonbo), CD45.2-PerCP-Cy5.5 (104, Tonbo), CD90.2-APC-Cy7 (30-H12, BioLegend), F4/80-APC-Cy7 (BM8, BioLegend), CD138-BV711 (281–2, BD), CD21-APC (B-ly4, BD), CD43-PE (S7, BD), GL7-eFluor660 (GL-7, eBioscience), CD23-eFluor450 (B3B4, eBioscience), IgM-FITC (II/41, eBioscience), Annexin V-APC (kit number 88–8007-72, eBioscience), and Zombie Yellow Fixable Viability Dye (kit number 423104, BioLegend). An LSRII was used for analysis and an ARIAII was used for sorting (BD Biosciences). All flow cytometry data was analyzed with FlowJo version 9.9.5. Preceding all flow cytometry analyses presented is the following gating strategy: 1) lymphocytes (forward scatter [FSC]-area by side scatter [SSC]-area), 2) singlets (FSC-width by FSC-height), 3) singlets (SSC-width by SSC-height), 4) live cells (viability dye⁻), 5) exclusion of non-B cell lineage cells (Thy1.1⁻F4/80⁻CD11b⁻).

Haines R.R., Barwick B.G., Scharer C.D., Majumder P., Randall T.D, & Boss J.M. (2018). The histone demethylase LSD1 regulates B cell proliferation and plasmablast differentiation. Journal of immunology (Baltimore, Md. : 1950), 201(9), 2799-2811.

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Phenotyping of B cell subsets

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For phenotyping of B cells, CD9⁺ and CD9⁻ B cells, the antibodies used were as follows: CD27-PE-Cy7 (clone 0323, Biolegend), CD38-PE (clone HB7, Biolegend), CD19-BV510 (clone HIB19, Biolegend), CD24-APC (clone SN3 A5-2H1D, eBioscience, San Diego, CA, USA), HLA-DR-PERCP (Clone L243, Biolegend), CD40-PE (Clone 5C3, BD Biosciences), CD86-PE (Clone 2331, BD Biosciences), CD25-PE/Cy5 (Clone M-A251, BD Biosciences), CD69-APC (Clone FN50, Biolegend), IgD-APC-Cy7 (Clone, IA6-2, Biolegend), IgM-FITC (Clone, SA-DA4, eBioscience), CD5-FITC (Clone, L17F12, eBioscience), CD10-APC (Clone, LT10, ImmunoTools, Friesoythe, Germany), CD20-PE-Dy647 (Clone, LT20, ImmunoTools) and CD21-FITC (Clone, LT21, ImmunoTools). Viability was assessed by 7AAD staining (BD Biosciences). All flow cytometric acquisition was performed with FACS Canto II, BD Biosciences. The data analysis was performed using FlowJo V7 (TreeStar Inc., Ashland, OR, USA). The gating strategy for the Bregs and single markers can be found in Supplementary Figure S1.

Mohd Jaya F.N., Garcia S.G., Borras F.E., Guerrero D., Chan G.C, & Franquesa M. (2021). In Vitro Characterization of Human CD24hiCD38hi Regulatory B Cells Shows CD9 Is Not a Stable Breg Cell Marker. International Journal of Molecular Sciences, 22(9), 4583.

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Comprehensive B Cell and Immune Profiling

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Flow-cytometric analysis or cell sorting of single cell suspensions generated from spleen or bone marrow was performed on an LSR-Fortessa or a FACS-Aria-III, respectively (both BD) and analyzed using FlowJo v10 software. Antibodies used (40 µl/sample): B220-FITC (biolegend RA3-6B2) 1:300, B220-APC/eF780 (ebioscience RA3-6B2) 1:200, B220-PE (BD RA3-6B2) 1:300, B220-PErCP-Cy5.5 (biolegend RA3-6B2) 1:300, CD19-BV605 (biolegend 6D5) 1:400, IgM-APC (biolegend RMM-1) 1:300, IgM-FITC (BD II/41) 1:300, IgM-eF450 (ebioscience eb121-15F9) 1:200, IgD-PerCP-Cy5.5 (biolegend 11-26c2a) 1:400, TCRβ-BV605 (BD H57-597) 1:200, TCRβ-FITC (ebioscience H57-597) 1:300, CD4-APC/Cy7 (BD GK1.5) 1:400, cKit-PE/Cy7 (biolegend 2B8) 1:300, cKit-PerCP-Cy5.5 (biolegend 2B8) 1:300, cKit-BV421 (biolegend 2B8) 1:300, Mac1-APC (ebioscience M1/70) 1:300, CD25-PE (biolegend PC61) 1:500, CD93-PE/Cy7 (ebioscience AA4.1) 1:300, NK1.1-APC (biolegend PK136) 1:300, AnnexinV-FITC (biolegend Lot: B206041) 1:1800, AnnexinV-eF450 (ebioscience; Lot: E11738-1633) 1:1000. FITC-F(ab’)₂ Fragment Goat Anti-Mouse IgM [1 μg/ml], μ Chain Specific (Jackson ImmunoResearch).

Schuler F., Weiss J.G., Lindner S.E., Lohmüller M., Herzog S., Spiegl S.F., Menke P., Geley S., Labi V, & Villunger A. (2017). Checkpoint kinase 1 is essential for normal B cell development and lymphomagenesis. Nature Communications, 8, 1697.

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Multicolor Flow Cytometry Antibody Panel

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The following antibodies were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated erythroid lineage cells (TER119; 561032), MAC1 (553310), GR1 (553127), CD11C (557400), B220 (553088), THY1.2 (553004), CD8A (553031), CD4 (553651), NK1.1 (553164), CD3ε (553062), CD19 (553785), TCRγδ (553177), phycoerythrin (PE)-conjugated SCA-1 (553336), CD4 (553653), CD19 (553786), GR-1 (553128), NK1.1 (553165), TCRβ (553172), allophycocyanin (APC)-conjugated LY5.1 (558701), LY5.2 (558702), C-KIT (553556), and CD19 (550992). FITC-IGM (11-5790-81) was purchased from eBioscience.

Ikawa T., Masuda K., Endo T.A., Endo M., Isono K., Koseki Y., Nakagawa R., Kometani K., Takano J., Agata Y., Katsura Y., Kurosaki T., Vidal M., Koseki H, & Kawamoto H. (2016). Conversion of T cells to B cells by inactivation of polycomb-mediated epigenetic suppression of the B-lineage program. Genes & Development, 30(22), 2475-2485.

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Multiparameter Flow Cytometry Immunophenotyping

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The surface markers included FITC‐CD3 (MA1‐7640, eBioscience, San Diego, CA, USA), APC‐Cy7‐CD4 (100413, Biolegend, San Diego, CA, USA), APC‐CD4 (100411, Biolegend), PerCP‐CD8 (100,731, Biolegend), PE‐Cy7‐CD8 (100721, Biolegend), PE‐Foxp3 (12‐5773‐82, eBioscience), APC‐Helios (17‐9883‐42, eBioscience), PerCP‐CD44 (103035, Biolegend), FITC‐CD62L (104405, Biolegend), FITC‐lin (22‐7770‐72, eBioscience), PerCP‐CD34 (50‐0341‐82, eBioscience), APC‐Sca‐1 (17‐5981‐82, eBioscience), PE‐Sca‐1 (12‐5981‐82, eBioscience), APC‐c‐kit (17‐1171‐82, eBioscience), PE‐c‐kit (12‐1171‐82, eBioscience), PE‐B220 (12‐0452‐82, eBioscience), FITC‐IgM (11‐5790‐81, eBioscience), PE‐Cy7‐CD19 (12‐0193‐82, eBioscience), APC‐CD19 (17‐0193‐82, eBioscience), PE‐Cy7‐CD16/32 (25‐0161‐82, eBioscience), PE‐Cy7‐CD45 (25‐0451‐82, eBioscience), PerCP‐5.5‐CD45.2 (45‐0454‐82, eBioscience), and APC‐Cy7‐CD45.1 (17‐0453‐82, eBioscience) obtained from Invitrogen. Intracellular staining with PE‐Foxp3 (72‐5775‐40, eBioscience) was performed with Foxp3 staining kits (Invitrogen, Carlsbad, CA, USA).

Chen J., Liu J, & Huang H. (2023). Lkb1 loss in regulatory T cells leads to dysregulation of hematopoietic stem cell expansion and differentiation in bone marrow. FEBS Open Bio, 13(2), 270-278.

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Immunophenotyping of Murine B Cells

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To analyze B cells in mice, blood cells and bone marrow cells were harvest and washed with PBS. Before staining, cells were blocked with 5% FBS in PBS for 30 min at 4 °C. And then cells were stained with membrane marker antibodies: PE-CD43, FITC-IgM, PE-B220 and PE/cy7-B220 purchased from eBioscience (USA). The control was performed that cells were stained with corresponding fluorescein conjugated isotype control antibody.

Shao W., Zhang C., Liu E., Zhang L., Ma J., Zhu Z., Gong X., Qin Z, & Qiu X. (2016). Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice. Scientific Reports, 6, 23669.

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