Anti mouse antibodies

Manufactured by BioLegend

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Anti-mouse antibodies are immunoglobulin molecules that specifically recognize and bind to mouse-derived proteins or other antigenic targets. These antibodies can be used in various immunological techniques to detect, quantify, or purify mouse-related biomolecules.

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Mouse anti-human monoclonal antibodies (4 citations) Soluble anti-mouse CD3 and CD28 antibodies (3 citations) Ultra-LEAF Purified anti-mouse Ly-6G antibodies (3 citations) Ter119-Alexa Fluor 647 anti-mouse antibodies (2 citations) Anti-mouse CD45-APC-Cy7 antibodies (2 citations) Mouse anti-EpCAM antibodies (2 citations) Anti-mouse iNOS antibodies (2 citations) BV421 conjugated anti-mouse CD25 antibodies (2 citations) FITC anti‐mouse FcεR1 antibodies (2 citations) TotalSeq-C anti-mouse Hashtag antibodies (2 citations)

22 protocols using anti mouse antibodies

Tumor Dissociation and Single-Cell Analysis

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Tumors were harvested, cut into small pieces with surgical scissors and sharp blade, and then digested in HBSS containing 0.5 mg/ml Collagenase D (Roche) and 40 µg/ml DNase I (Roche) in a 37°C shaker for 20–30 min. Digestion was stopped by adding 0.5 mg/ml EDTA in HBSS, and single-cell suspensions were prepared for antibody staining. The following anti-mouse antibodies obtained from BioLegend were used for the staining and analysis: anti-CD45 (30-F11), anti-CD3 (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD44 (IM7), anti-FoxP3 (MF-14), anti-NK1.1 (PK136), anti-CD11b (M1/70), anti-IFNγ (XMG1.2), anti-TNFα (TN3-19.12), and anti-GzmB (GB11). Intracellular staining was performed using the eBioscience Intracellular Fixation and Permeabilization Buffer Set (88-8824-00). Dead cells were excluded using 7-aminoactinomycin D staining. The samples were run on a BD LSRII flow cytometer, and data were analyzed using FlowJo software.

Jiang X., Muthusamy V., Fedorova O., Kong Y., Kim D.J., Bosenberg M., Pyle A.M, & Iwasaki A. (2019). Intratumoral delivery of RIG-I agonist SLR14 induces robust antitumor responses. The Journal of Experimental Medicine, 216(12), 2854-2868.

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Multicolor Flow Cytometry Immunophenotyping

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Lamina propria lymphocytes were stained with antibodies for 30 min at 4°C. The following anti-mouse antibodies (all from Biolegend) were used: Alexa Fluor 700-CD45 (Clone 30-F11), APC/Cyanine7-F4/80 (BM8), PerCP-CD11b (M1/70), Brilliant Violet 570-Ly6G (1A8), Brilliant Violet 421-CD86 (GL-1), FITC-CD206 (C068C2), PE-Ly6C (HK1.4), PerCP/Cyanine5.5-CD3 (17A2), BV421-CD4 (GK1.5), PerCP-CD8a (53–6.7) and FITC-B220 (RA3-6B2). All data were acquired on the Gallios instrument (Beckman) and analyzed using FlowJo X (TreeStar) or Beckman analysis software.

Liu Y., Xu J., Ren X., Zhang Y., Ke Z., Zhou J., Wang Y., Zhang Y, & Liu Y. (2022). Cholecystectomy-induced secondary bile acids accumulation ameliorates colitis through inhibiting monocyte/macrophage recruitment. Gut Microbes, 14(1), 2107387.

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Multiparameter Flow Cytometry Analysis

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Cell suspensions were labeled directly with the following fluorochrome-conjugated anti-mouse antibodies purchased from either BioLegend or eBioscience: CD4 (RM4-5), CD8α (53–6.7), CD24 (M1/69), CD25 (PC61.5), CD73 (TY/11.8), and anti-human CD2 (RPA-2.10). To block the Fc-receptors, the staining mix always contained unconjugated anti-FcRγIII/II antibody (BioXcell; final concentration 10 μg ml⁻¹). For exclusion of dead cells, 4′,6-Diamidine-2′-phenylindole dihydrochloride (Merck) was used. Stained cells were assessed by LSRFortessa™ or FACSCanto™ II flow cytometer (BD Biosciences) and data were analyzed with FlowJo^® software (TreeStar).

Herppich S., Toker A., Pietzsch B., Kitagawa Y., Ohkura N., Miyao T., Floess S., Hori S., Sakaguchi S, & Huehn J. (2019). Dynamic Imprinting of the Treg Cell-Specific Epigenetic Signature in Developing Thymic Regulatory T Cells. Frontiers in Immunology, 10, 2382.

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Flow Cytometry Analysis of Splenic and Blood Cells

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Flow cytometry analysis was performed using a Guava 8HT (EMD Millipore Corporation, Billerica, MA USA). Splenic and blood cells from control and tolerant mice were collected and labeled with antimouse antibodies (BioLegend, San Diego, CA) for cell surface markers: CD4-PE and CD25- APC/Cy7. After cell membrane permeabilization with saponin, cells were intracellular labeled with antimouse antibodies: FOXP3-PE/Cy7, IL-17a- APC and IL-6-FITC (BioLegend).

Andrade M.M., Ariga S.S., Barbeiro D.F., Barbeiro H.V., Pimentel R.N., Petroni R.C, & Soriano F.G. (2019). Endotoxin tolerance modulates TREG and TH17 lymphocytes protecting septic mice. Oncotarget, 10(37), 3451-3461.

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Splenocyte Isolation and Immune Cell Analysis

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All splenocytes were flushed out from spleen by 1× PBS and then passed through a 200-mesh sieve to obtain single cell suspension. For flow cytometric detection, the following anti-mouse antibodies (all from Biolegend, San Diego, CA, US) were used: F4/80-APC, CD11b-FITC, Ly6G-PE, Ly6C-APC, Gr-1-FITC. Cells were detected using a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Data were analyzed using FlowJo software (Treestar, Inc., San Carlos, CA).

Qu J., Sun Z., Peng C., Li D., Yan W., Xu Z., Hou Y., Shen S., Chen P, & Wang T. (2021). C. tropicalis promotes chemotherapy resistance in colon cancer through increasing lactate production to regulate the mismatch repair system. International Journal of Biological Sciences, 17(11), 2756-2769.

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Multiparametric Flow Cytometry of SVF Cells

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Purified SVF cells were blocked with anti-mouse CD16/32 antibody for 10 minutes on ice. For analysis of immune cell populations, cells were further incubated for 30 minutes on ice in the dark with the following anti-mouse antibodies obtained from BioLegend: APC-CD11b, APC-Cy7-CD11c, PE-CD206 or PE/Cy7-CD206, PerCP/Cy5.5-Ly6G, FITC-CD3, PerCP/Cy5.5-CD19, APC/Cy7-NK1.1. After antibody conjugation, cells were washed with Cell Staining Buffer and analyzed on a Becton-Dickinson LSRII analytical flow cytometer. For analysis of intracellular IL-6, purified SVF cells obtained by pooling the SVF of two AL mice were incubated with 1× Brefeldin A solution (BioLegend) for 4h in culture medium at 37°C. Blocking and cell surface staining were performed as described above followed by fixation and permeabilization using Fixation/Permeabilization Reagents and the protocol recommended by the manufacturer (eBioscience, San Diego, CA). Intracellular staining of IL-6 was performed using anti-mouse PE-IL-6 and negative control (PE-Rat IgG1, κ Isotype control). Cells were analyzed using iCyt Synergy Cell Sorter (Sony Biotechnology San Jose, CA). Analysis of flow cytometry data was performed using FlowJo data analysis software (FlowJo, LLC, Ashland, OR).

Starr M.E., MSteele A., Cohen D.A, & Saito H. (2016). Short-Term Dietary Restriction Rescues Mice From Lethalabdominal Sepsis and Endotoxemia, and Reduces the Inflammatory/Coagulant Potential of Adipose Tissue. Critical care medicine, 44(7), e509-e519.

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FACS Analysis of Immune Cell Subsets

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Anti-mouse antibodies (BioLegend unless otherwise noted) used for FACS analysis were as follows: FITC-conjugated anti-CD3 (17A2) and anti-CD4 (GK1.5; BD Pharmingen); PE-conjugated anti-CD4 (GK1.5), anti-CD19 (6D5), anti-CD69 (H1.2F3; BD Pharmingen), anti-IFN-γ (XMG1.2; eBioscience), anti-TNF-α (MP6-XT22; eBioscience), and anti-IL-2 (JES6-5H4; eBioscience); PE-Cy7-conjugated anti-NK-1.1 (PK136) and anti-CD44 (IM7; BD Pharmingen); APC-conjugated anti-IFN-γ (XMG1.2), anti-TNF-α (MP6-XT22) and anti-IL-7Rα (A7R34), and BV421-conjugated anti-CD62L (MEL-14).

Clarke D., Letendre C., Lecours M.P., Lemire P., Galbas T., Thibodeau J, & Segura M. (2016). Group B Streptococcus Induces a Robust IFN-γ Response by CD4+ T Cells in an In Vitro and In Vivo Model. Journal of Immunology Research, 2016, 5290604.

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Murine Hematopoietic Cell Staining

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BM or spleen single-cell suspensions were prepared as previously described (Chan et al., 2011 (link)) and stained with combinations of the following anti–mouse antibodies (BioLegend, unless otherwise stated): CD11b(Mac1) (FITC-conjugated; SouthernBiotech), CD4 (phycoerythrin [PE] conjugated), Ly-6G(Gr-1) (PE-Cy7 conjugated), CD45R(B220) (allophycocyanin [APC]-conjugated; Invitrogen), CD117(c-kit) (PE-Cy7 conjugated), Ly-6A/E(Sca-1) (Pacific Blue [PB] conjugated), CD135(Flt3) (PE conjugated), CD34 (FITC conjugated; BD), CD127(IL-7Ra) (PE conjugated), CD16/32(FcRγ) (PE conjugated), and Mouse Lineage antibody cocktail (APC conjugated; BD). LT-HSC, ST-HSC, MPP, CMP, GMP, and MEP were defined as previously described (Chan et al., 2011 (link)). All analyses considered only 7-AAD⁻ (BD) populations. Annexin V (APC conjugated; BD) and 7-AAD were used in cell viability assays according to the manufacturer’s protocol. Flow cytometry was performed on a CyAn ADP FlowCytometer (Dako) or a BD LSRFortessa cell analyzer and all data were analyzed with FlowJo software (Tree Star).

Giotopoulos G., van der Weyden L., Osaki H., Rust A.G., Gallipoli P., Meduri E., Horton S.J., Chan W.I., Foster D., Prinjha R.K., Pimanda J.E., Tenen D.G., Vassiliou G.S., Koschmieder S., Adams D.J, & Huntly B.J. (2015). A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression. The Journal of Experimental Medicine, 212(10), 1551-1569.

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Murine AdMSCs Characterization by Flow

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Murine AdMSCs were characterized by flow cytometry. Three different pools of mAdMSCs (each of them isolated from 10 to 15 mice) and at different culture passages (from passage 2 up to passage 5) were analyzed the same day, and at least 5 × 10⁴ events for all samples were recorded. Briefly, cells were detached with TrypLE Express dissociation reagent (Gibco), washed, and resuspended in PBS (Gibco) containing 1% fetal bovine serum (Lonza). Then, mAdMSCs were incubated with fluorochrome-conjugated anti-mouse antibodies specific for the surface markers CD73, CD90, CD105, CD44, PSGL-1, CD43, CD29, Sca-1, CD106, CD166, CD34, CD45, CXCR4, c-Kit, CD80, CD86 (all from BioLegend, San Diego, CA, United States) and for the tetrasaccharide sialyl Lewis X/A (clone HECA452; BD Biosciences), or their specific control isotype antibodies, for 30 min in the dark at 4°C. Finally, cells were washed, resuspended at 1 × 10⁶ cells/ml and analyzed in a FACSCanto II flow cytometer (BD Biosciences).

García-Bernal D., García-Arranz M., García-Guillén A.I., García-Hernández A.M., Blanquer M., García-Olmo D., Sackstein R., Moraleda J.M, & Zapata A.G. (2020). Exofucosylation of Adipose Mesenchymal Stromal Cells Alters Their Secretome Profile. Frontiers in Cell and Developmental Biology, 8, 584074.

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Phenotypic Characterization of Antigen-Specific T Cells

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Mouse splenocytes were stimulated at 37 °C and 5% CO₂ for 5 h with relevant peptide pools at 1 ng/µL (test of Ad-Ii-SIVCErvv, homologous prime-boost) or 0.67 ng/µL (test of Ad-HIVB, heterologous prime-boost) and 3 µM Monensin. Peptides were obtained from the NARP and the SIV CE peptide pool was constructed as described [11 (link)]. Subsequently, cells were stained according to standard protocols [23 (link), 24 (link)] using anti-mouse antibodies (Biolegend): PerCP/Cy5.5-CD8, FITC-CD4, Pacific Blue™-B220, APC/Cy7-CD44, APC-IFNγ, PE/Cy7-TNFα. Flow cytometry was performed in an LSRII instrument (BD Biosciences) and the data analyzed with FlowJo 10 software (Tree Star, Ashland, OR) using the gating strategy shown in Additional file 1.

Schwerdtfeger M., Andersson A.M., Neukirch L, & Holst P.J. (2019). Virus-like vaccines against HIV/SIV synergize with a subdominant antigen T cell vaccine. Journal of Translational Medicine, 17, 175.

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