Efluor 450 f4 80

Manufactured by Thermo Fisher Scientific

EFluor 450-F4/80 is a fluorescent-conjugated antibody for detection and analysis of the mouse macrophage surface marker F4/80 using flow cytometry. The EFluor 450 fluorophore emits blue light, allowing for multicolor analysis of samples.

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F4/80 (353 citations) EFluor 450 (209 citations) EFluor 450-anti-F4/80 (BM8, (2 citations) EFluor 450-labeled F4/80 (BM8) (2 citations) Anti-mouse F4/80-eFluor 450 antibody (2 citations)

3 protocols using efluor 450 f4 80

Fluorescent Antibody Flow Cytometry

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Fluorescently labeled mAbs for flow cytometry were used as follows: FITC-B220, eFluor 450-F4/80, APC eFluor 780-CD8, Alexa Fluor 700-CD4, APC-Foxp3, and PerCP-Cy5.5-CD44 (eBioScience), and Foxp3/Transcription Factor Fixation/Permeabilization (eBioscience, 501129060). Unlabeled TCR Cβ-specific H57-597 was expressed and purified from the B-cell hybridoma (a gift from Kappler/Marrack lab). All peptides were synthesized by Peptide 2.0 Inc.

Li W., Li R., Wang Y., Zhang Y., Tomar M.S, & Dai S. (2022). Calcitonin gene-related peptide is a potential autoantigen for CD4 T cells in type 1 diabetes. Frontiers in Immunology, 13, 951281.

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Multiparameter Flow Cytometry Analysis

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After tetramers staining, the cells were stained with surface fluorescentrly labeled mAbs as follows: FITC-B220, eFluor 450-F4/80, APC eFluor 780-CD8, Alexa Fluor 700-CD4⁺, and PerCP-Cy5.5-CD44 (eBioScience). After MHC II- peptide tetramer staining and cell surface staining, cells were fixed with 4% paraformaldehyde (Thermo Scientific Cat: 00-5523, CA, US.). Intracellular staining with Foxp3-APC was done using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Cat: 00-5523, CA, US.) according to the manufacturer’s instructions. Stained single-cell suspensions were analyzed using a Fortessa flow cytometer running FACSDiva (BD Biosciences, US). Flow Cytometry Data File Standard Version 3.0 (FSC 3.0) files were analyzed with Flowjo 10.0 Software.

Li W., Zhang Y., Li R., Wang Y., Chen L, & Dai S. (2022). A Novel Tolerogenic Antibody Targeting Disulfide-Modified Autoantigen Effectively Prevents Type 1 Diabetes in NOD Mice. Frontiers in Immunology, 13, 877022.

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Isolation and Immunophenotyping of Splenocytes

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Splenocytes were isolated from spleens of bone marrow chimeric mice by homogenization on 70 μm mesh cell strainers on tissue culture plates, followed by washing in RPMI media and incubating in ACK RBC lysis buffer (Thermo Fisher Scientific) for 4 minutes at 37°C to remove red blood cells. Cells were incubated with NIR Zombie (1:200, Biolegend) in PBS for live/dead staining for 15 minutes at room temperature, followed by PE CD45.1 (1:200, clone A20, eBioscience), APC CD45.2 (1:200, clone 104, Biolegend), BV 605 CD3e (1:200, clone 145–2C11, Biolegend), BV 650 CD19 (1:200, clone 6D5, Biolegend), FITC CD11b (1:100, clone M1/70, eBioscience), Percp Cy5.5 Ly6G/Gr-1 (1:200, clone RB6–8C5, eBioscience), eFluor 450 F4/80 (1:200, clone BM8, eBioscience) and Fc blocker (1:100, anti-mouse CD16/32,BD) at 4°C in the dark for 20 minutes, and washed in PBS/0.5% BSA. Analysis was performed using an LSRII (Becton Dickinson) flow cytometer and FlowJo. v10 (Treestar).

Han C.Y., Kang I., Harten I.A., Gebe J.A., Chan C.K., Omer M., Alonge K.M., den Hartigh L.J., Kjerulf D.G., Goodspeed L., Subramanian S., Wang S., Kim F., Birk D.E., Wight T.N, & Chait A. (2020). Adipocyte-Derived Versican and Macrophage-Derived Biglycan Control Adipose Tissue Inflammation in Obesity. Cell reports, 31(13), 107818.

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