Anti mouse cd11b efluor450

Manufactured by Thermo Fisher Scientific

The Anti-mouse CD11b-eFluor450 is a fluorochrome-conjugated antibody that binds to the CD11b antigen expressed on the surface of mouse myeloid cells, including monocytes, macrophages, and granulocytes. It is designed for use in flow cytometry applications to identify and analyze these cell populations.

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Anti-CD11b-eFluor450 CD11b-eFluor450 Anti-CD11b CD11b

6 protocols using anti mouse cd11b efluor450

Phagocytosis of Stressed Neutrophils

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Lithium-heparinized blood of BALB/c WT and Ncf1** mice was collected. To evaluate whether platelets or complement affect phagocytosis of SNECs samples were centrifuged at 1000 rpm for 5 min to separate the plasma from the whole blood. The plasma was carefully removed and incubated with 1:1000 Prostaglandin E1 (Sigma) and 1:250 Apyrase (Sigma). Plasma was then used without further treatment or depleted of platelets via centrifugation at 400g and collection of the supernatants. To further deplete complement, platelet free plasma was heat inactivated via incubation in a water bath (57 °C, 30min). Pellets and plasma were checked for platelets via flow cytometry (FSC/SSC and CD61-PE). Prior to adding pHrodo-labelled SNECs samples were reconstituted with either plasma, platelet depleted plasma, or platelet-depleted and heat-inactivated plasma and incubate at 37 °C for 4 h. Samples were then stained with anti-mouse CD11b-eFluor450 (eBioscience) and anti-mouse Ly6C-APC (BioLegend) for 20min on ice, followed by hypotonic water lysis of erythrocytes and determination of phagocytosis indices as described before.

Hahn J., Euler M., Kilgus E., Kienhöfer D., Stoof J., Knopf J., Hahn M., Harrer T., Hultqvist M., Olofsson P., Mokhir A., Holmdahl R., Herrmann M., Schett G., Muñoz L.E, & Hoffmann M.H. (2019). NOX2 mediates quiescent handling of dead cell remnants in phagocytes. Redox Biology, 26, 101279.

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Lung Cell Population Characterization

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The adipose tissues and bronchus of each group of lungs were removed. The lungs were then cut into pieces and digested in RPMI 1640 medium (VivaCell, Shanghai, China) containing 10% FBS (Sperikon Life Science & Biotechnology Co., Ltd., Chengdu, China) and collagenase VIII (250 U/mL; Sigma Aldrich, Saint Louis, MO, USA) at 37 °C for 30 min. Cell surface staining was performed by incubating the cells with the indicated antibodies on ice for 15 min. Cells were washed and analyzed on an LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) using FlowJo software (BD Biosciences, San Diego, CA, USA).
To measure the percentages of CD4⁺ and CD8⁺ cells among CD3⁺ mononuclear cells, the cells were incubated with anti-mouse CD3e-FITC (1:1200, eBioscience); anti-mouse CD4-APC (1:400, eBioscience); anti-mouse CD8a-PE (1:1600, BioLegend); anti-mouse CD45R-PerCP-Cyanine5.5 (1:800, eBioscience) for 20 min at room temperature.
To measure the percentages of macrophages and dendritic cells, cells were incubated with anti-mouse I-A/I-E-PE/Cyanine7 (1:3200, BioLegend); anti-mouse F4/80-FITC (1:100, eBioscience); anti-mouse CD11b-eFluor™ 450 (1:800, eBioscience); anti-mouse CD11c-PerCP-Cyanine5.5 (1:200, eBioscience); anti-mouse Ly-6C-APC (1:800, eBioscience) for 20 min at room temperature.

Chen J., Wang X., Li J., Sun L., Chen X., Chu Z., Zhang Z., Wu H., Zhao X., Li H, & Zhang X. (2023). Influenza A Virus Weakens the Immune Response of Mice to Toxoplasma gondii, Thereby Aggravating T. gondii Infection. Veterinary Sciences, 10(5), 354.

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Bone Marrow Immune Cell Profiling

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RBCs in BM cells from mice were lysed using red cell lysis buffer (Thermo Fisher Scientific, 89900) at room temperature for 4 minutes and then washed with PBS (containing 1% BSA) 2 times. For LKS, HSC, and MPP staining, APC mouse lineage antibody cocktail (BD Biosciences, 558074); anti-CD117 (c-kit) rat monoclonal antibody (APC/Cy7) (BioLegend, 105825); Ly-6A/E (Sca-1) monoclonal antibody (D7) (eBioscience, 25-5981-81); PE-Cyanine7 CD150 monoclonal antibody (mShad150), eFluor 450 (eBioscience, 48-1502-80); CD48 monoclonal antibody (HM48-1); PerCP-eFluor 710 (eBioscience, 46-0481-80); PE rat anti–mouse CD135 (BD Biosciences, 561068); CD34 monoclonal antibody (RAM34); and FITC (eBioscience, 11-0341-81) were added to BM cells and were incubated together on ice for 30 minutes. For myeloid cells including monocyte, macrophage, DC, and monocyte staining, APC anti–mouse CD115 (CSF-1R) antibody (BioLegend, 135509); anti–Ly-6C rat monoclonal antibody (Brilliant Violet 605, HK1.4) (BioLegend, 128035); PE/Cy7 anti–mouse Ly-6G Antibody [1A8] (BioLegend, 127617); CD11c armenian hamster anti–mouse Alexa Fluor 700 (Thermo Fisher Scientific, 56-0114-80); anti–mouse CD11b eFluor 450 (eBioscience, 48-0112-80); F4/80 monoclonal antibody (BM8); and Alexa Fluor 488 (BioLegend, 123120) were added to BM cells and were then incubated together on ice for 30 minutes.

Zhang B.Z., Shuai H., Gong H.R., Hu J.C., Yan B., Yuen T.T., Hu Y.F., Yoon C., Wang X.L., Hou Y., Lin X., Huang X., Li R., Au-Yeung Y.M., Li W., Hu B., Chai Y., Yue M., Cai J.P., Ling G.S., Hung I.F., Yuen K.Y., Chan J.F., Huang J.D, & Chu H. (2022). Bacillus Calmette-Guérin–induced trained immunity protects against SARS-CoV-2 challenge in K18-hACE2 mice. JCI Insight, 7(11), e157393.

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Isolation and Characterization of Infiltrating Lymphocytes from Islets

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Handpicked islets were pooled in 24-well plates for 24 h in RPMI supplemented with 10% FCS, penicillin–streptomycin, and 5.10⁻⁵ M β-mercaptoethanol allowing extrusion of infiltrating lymphocytes (25 (link)). Infiltrating lymphocytes were recovered. Cells recovered were used for the analysis of T cells and antigen-presenting cells. T-cell analysis was performed using the Foxp3 intra-staining buffer set according to the recommendation of the manufacturer (eBioscience) with the following combination: anti-mouse CD45-efluo450, anti-mouse CD3ϵ-AlexaFluo700, anti-mouse B220-HorizonViolet500, anti-mouse CD8α-Percp-Cy5.5, anti-mouse CD4-PE, anti-mouse Foxp3-APC, and anti-mouse CD25-BrillantViolet711 mAbs (eBioscience). β Cells and dendritic cells were analyzed with anti-mouse CD45-AlexaFluo700, anti-mouse TCRβ-APC-Cy7, anti-mouse B220-HorizonViolet500, anti-mouse CD11b-efluor450, anti-mouse CD11c-APC, anti-mouse CD8α-BrillantViolet605, and anti-mouse CD4-PE mAbs (eBioscience). Acquisitions were performed with a BD LSR-Fortessa flow cytometer and analyzed using FlowJo 10.7.1 software.

Luce S., Guinoiseau S., Gadault A., Letourneur F., Nitschke P., Bras M., Vidaud M., Charneau P., Larger E., Colli M.L., Eizirik D.L., Lemonnier F, & Boitard C. (2021). A Humanized Mouse Strain That Develops Spontaneously Immune-Mediated Diabetes. Frontiers in Immunology, 12, 748679.

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Phagocytosis of SNECs by Mouse Leukocytes

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Lithium-heparinized blood of BALB/c WT and Ncf1** mice was collected. To determine whether plasma factors affected phagocytosis of SNECs samples were centrifuged at 1000 rpm for 5 min to separate the plasma from the whole blood. Then the plasma was carefully removed and pooled Ncf1** plasma was added to WT cells and vice versa. As a control, cells were also incubated with pooled autologous plasma from the same genotype. Samples were then mixed with pHrodo-labelled SNECs and incubated at 37 °C for up to 4 h. Samples were then stained with anti-mouse CD11b-eFluor450 (eBioscience) and anti-mouse Ly6C-APC (BioLegend) for 20min on ice, followed by hypotonic water lysis of erythrocytes and analysis using a Beckman Coulter Gallios™. The PhIx was then calculated as previously described.

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Phagocytosis Assay for Neutrophils

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Lithium-heparinized blood from BALB/c WT and Ncf1** mice or human NHD and SLE/CGD patients was mixed with SNECs in a 1:2 ratio and incubated at 37 °C. In some experiments, the blood was preincubated with varying concentrations of H₂O₂ (10 min), of the aminoferrocene compound MIS43 (10 min) [32 (link)], varying concentrations of catalase (Sigma) or butylated hydroxyanisole (BHA, Sigma) for 15 min before adding SNECs. Cells were stained with anti-mouse CD11b-eFluor450 (eBioscience), anti-mouse Ly6C-APC (BioLegend), Ly6G-PE/Cy7 (BioLegend), CCR2-FITC (BioLegend), I-A/I-E (MHC II)-Alexa Fluor 700 (BioLegend) or anti-human CD14-eFluor450 (eBioscience), CD16-APC/Cy7 and CCR2-PerCP/Cy5.5 (BioLegend), respectively. Samples were subjected to hypotonic water lysis of erythrocytes and analyzed using a Beckman Coulter Gallios™ flow cytometer. The Phagocytosis index (PhIx) was calculated in the following way: PhIx = (%PI/pHrodo-positive phagocytes x MFI^PI/pHrodo). For the analysis of cytokine/chemokine production upon phagocytosis, plasma was isolated from blood incubated with SNECs (3 h) and subjected to LegendPlex bead-based assay (BioLegend).

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