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Igm 2 41

Manufactured by Thermo Fisher Scientific
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The IgM (II/41) is a lab equipment product offered by Thermo Fisher Scientific. It is a reagent designed for the detection and quantification of IgM antibodies in biological samples. The core function of this product is to provide a tool for researchers and clinicians to analyze IgM levels, which can be relevant for various immunological and diagnostic applications.

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Lab products found in correlation

Cd5 53 7.3, by Thermo Fisher Scientific (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Peanut agglutinin, by Vector Laboratories (2 mentions) Axio imager m1 microscope, by Zeiss (2 mentions) Metallophilic macrophages 1 moma 1, by Abcam (2 mentions) Cd35 8c12, by BD (2 mentions) Ec plan neofluar objective, by Zeiss (2 mentions) B220 ra3 6b2, by Thermo Fisher Scientific (2 mentions) Cy3 streptavidin, by Jackson ImmunoResearch (2 mentions) Cd3e 145 2c11, by BD (2 mentions) Tissue tek oct, by Sakura Finetek (2 mentions) Cd4 gk1, by Thermo Fisher Scientific (2 mentions) Igd 11 26c 2a, by BioLegend (2 mentions) Mwreg30, by Thermo Fisher Scientific (1 mentions) Cd11c n418, by Thermo Fisher Scientific (1 mentions) Cd11b m1 70, by Thermo Fisher Scientific (1 mentions) Cd3ε 145 2c11, by BioLegend (1 mentions) Ly6c hk1, by BioLegend (1 mentions) Gr 1 rb6 8c5, by BioLegend (1 mentions) C kit 2b8, by BioLegend (1 mentions)

6 protocols using igm 2 41

1

Identification of B Cell Subsets by Flow Cytometry

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Single-cell suspensions from lungs and spleen were depleted of red blood cells using hypotonic lysis. Cells were resuspended in phosphate buffered saline (PBS) with 0.2% bovine serum albumin (BSA) and 0.1% sodium azide (FACS buffer). Single cell suspensions were incubated for 15 min with anti-CD16/32 (Fc block), and stained for 30 min at 4°C. DAPI, anti-mouse CD3 (17A2), CD19 (1D3), CD11b (M1/70), CD5 (53-7.3), and IgM (II/41) were purchased from eBioscience. Cells were analyzed on LSRII (Becton Dickinson). More than 0.5 × 105 cells were analyzed per sample, with dead cells excluded by DAPI positive staining. Surface marker analysis was performed using FlowJo software (Treestar Inc.). B cells (CD19+), B-1 B cells (CD19+, sIgM+, CD11b+), B-1a B cells (CD19+, sIgM+, CD11b+, CD5+), and B-1b B cells (CD19+, sIgM+, CD11b+, CD5−) were identified with the appropriate gating. For gating strategy and controls see Figure 1.
Baldan A., Gonen A., Choung C., Que X., Marquart T.J., Hernandez I., Bjorkhem I., Ford D.A., Witztum J.L, & Tarling E.J. (2014). ABCG1 is required for pulmonary B-1 B cell and natural antibody homeostasis. Journal of immunology (Baltimore, Md. : 1950), 193(11), 5637-5648.
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2

Multi-Parameter Cell Sorting and Analysis

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For cell sorting and analysis, monoclonal antibodies to CD41 (MWReg30, eBioscience), CXCR4 (2B11, eBioscience), CD11b (M1/70, eBioscience), F4/80 (BM8, eBioscience), Gr-1 (RB6-8C5, Biolegend), Ly6C (HK1.4, Biolegend), CD11c (N418, eBioscience), CD45.1 (A20, eBioscience), CD45.2 (104, Biolegend), CD4 (GK1.5, eBioscience), CD8 (53–6.7, Biolegend), INF-γ (XMG1.2, Biolegend), IL4 (11B11, Biolegend), CD34 (RAM34, eBioscience), Sca-1 (D7, Biolegend), c-kit (2B8, Biolegend), CD135 (A2F10, Biolegend), CD3ε (145–2 C11, Biolegend), CD45R (RA3-6B2, Biolegend), TER-119 (Ter-119, Biolegend), IgM (II/41, eBioscience), FγRII (93, Biolegend), IL-7R (A7R34, Biolegend), TNFα (MP6-XT22, Invitrogen), IL-6 (MP5-20F3, Biolegend), OVA257-264 (SIINFEKL) peptide bound to H-2Kb (eBio25-D1.16 (25-D1.16), Invitrogen) and IL-2 (JES6-5H4, eBioscience) were used where indicated.
Wang J., Xie J., Wang D., Han X., Chen M., Shi G., Jiang L, & Zhao M. (2022). CXCR4high megakaryocytes regulate host-defense immunity against bacterial pathogens. eLife, 11, e78662.
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3

Immunohistochemical Analysis of Splenic Architecture

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Spleens were embedded in Tissue Tek O.C.T (Sakura Finetek) and frozen at –80°C. Sections were fixed with acetone, blocked with PBS+5% FBS for 1 h at 19–25 °C and stained with a combination of various antibodies: Peanut agglutinin (Vector Labs), B220 (RA3-6B2, eBioscience), Metallophilic Macrophages -1 (Moma-1, Abcam), IgM (II/41, eBioscience), CD5 (53-7.3, eBioscience), CD35 (8C12, BD Pharmingen), CD3e (145-2C11) for 2 h at 19–25 °C. Streptavidin Cy3 (Jackson) was used in a second step to detect biotinylated antibodies. Sections were washed 3 times in between every step with PBS+0.5% Tween. Images were acquired on a Zeiss Axio ImagerM1 Microscope (Zeiss) using SlideBook software (Intelligent Imaging Innovations) at 19–25 °C with EC Plan-NEOFLUAR objectives (Zeiss).
Jellusova J., Cato M.H., Apgar J.R., Ramezani-Rad P., Leung C., Chen C., Richardson A.D., Conner E.M., Benschop R.J., Woodgett J.R, & Rickert R.C. (2017). GSK3 is a metabolic checkpoint regulator in B cells. Nature immunology, 18(3), 303-312.
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4

Immunohistochemical Analysis of Splenic Architecture

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Spleens were embedded in Tissue Tek O.C.T (Sakura Finetek) and frozen at –80°C. Sections were fixed with acetone, blocked with PBS+5% FBS for 1 h at 19–25 °C and stained with a combination of various antibodies: Peanut agglutinin (Vector Labs), B220 (RA3-6B2, eBioscience), Metallophilic Macrophages -1 (Moma-1, Abcam), IgM (II/41, eBioscience), CD5 (53-7.3, eBioscience), CD35 (8C12, BD Pharmingen), CD3e (145-2C11) for 2 h at 19–25 °C. Streptavidin Cy3 (Jackson) was used in a second step to detect biotinylated antibodies. Sections were washed 3 times in between every step with PBS+0.5% Tween. Images were acquired on a Zeiss Axio ImagerM1 Microscope (Zeiss) using SlideBook software (Intelligent Imaging Innovations) at 19–25 °C with EC Plan-NEOFLUAR objectives (Zeiss).
Jellusova J., Cato M.H., Apgar J.R., Ramezani-Rad P., Leung C., Chen C., Richardson A.D., Conner E.M., Benschop R.J., Woodgett J.R, & Rickert R.C. (2017). GSK3 is a metabolic checkpoint regulator in B cells. Nature immunology, 18(3), 303-312.
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5

Multiparametric Analysis of Immune Cells

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Cell suspensions prepared as described above were blocked with CD16/CD32 (Mouse BD Fc Block, 2.4G2, BD Biosciences). For B cell compartment analysis, suspensions were stained with antibodies against CD45 (30-F11, Tonbo), CD4 (GK1.5, eBioscience), CD19 (6D5, Biolegend), IgD (11–26c.2a, Biolegend), IgG (polyclonal, Jackson ImmunoResearch), IgM (II/41, Thermofisher), CD95 (DX2, Biolegend), and GL7 antigen (GL7, Biolegend). Dead cells were excluded with eBioscience Fixable Viability Dye eFluor 455UV (Thermofisher). In phagocyte depletion experiments, suspensions were stained with antibodies against CD45 (30-F11, Tonbo), I-A/I-E a (M5/114.15.2, Biolegend), CD11c (N418, Biolegend), CD11b (M1/70, Thermofisher), CX3CR1 (SA011F11, Biolegend), and CD103 (2E7, Thermofisher). Dead cells were excluded with eBioscience Fixable Viability Dye eFluor 506 (Thermofisher). Bacterial FISH staining was performed with Cy5-labeled EUB338 5’-GCTGCCTCCCGTAGGAGT-3’ probe (Integrated DNA Technologies; Table S1). Images were acquired under an inverted Nikon Eclipse Ti microscope (Nikon). Flow cytometry was performed using a LSRFortessa (BD Biosciences) and data were analyzed with FlowJo software (TreeStar).
Doron I., Leonardi I., Li X.V., Fiers W.D., Semon A., Bialt-DeCelie M., Migaud M., Gao I.H., Lin W.Y., Kusakabe T., Puel A, & Iliev I.D. (2021). Human gut mycobiota tune immunity via CARD9-dependent induction of anti-fungal IgG antibodies. Cell, 184(4), 1017-1031.e14.
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6

Confocal Imaging of Splenic Immune Cells

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Confocal imaging was performed on spleen sections. The following antibodies were used: IgM (II/41; Thermo Fischer Scientific), IgD (11-26.c2a; BioLegend), CD1d (1B1; eBioscience), CD169 (MOMA-1; Abcam). Briefly, 8-μm spleen frozen sections were fixed for 10 min in cold acetone. After washing with PBS, sections were blocked with 10% BSA or Streptavidin/Biotin Blocking kit (Vector) in the case of the MOMA-1 biotinylated antibody. After washing with PBS, sections were stained with the primary antibodies ON at 4°C, followed by a 60-min incubation period with Streptavidin-BV-421 (BioLegend) for MOMA-1 antibody. Sections were mounted with ProLong Gold antifade reagent (Thermo Fisher Scientific) and images were acquired on a Zeiss LSM780 confocal microscope equipped with 488-, 561-, and 633-nm lasers. Images were analyzed with Imaris software.
Andreani V., Ramamoorthy S., Fässler R, & Grosschedl R. (2022). Integrin β1 regulates marginal zone B cell differentiation and PI3K signaling. The Journal of Experimental Medicine, 220(1), e20220342.
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