Cd64 pe cy7

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CD64-PE/Cy7 is a fluorescently-labeled antibody that binds to the CD64 cell surface antigen. CD64, also known as the high-affinity immunoglobulin gamma Fc receptor I (FcγRI), is a receptor that plays a role in phagocytosis and antibody-dependent cell-mediated cytotoxicity. The PE/Cy7 fluorophore combination provides a bright signal and allows for multicolor flow cytometry applications.

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7 protocols using cd64 pe cy7

Comprehensive Flow Cytometric Analysis of Murine Lung Cells

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In brief, lungs were isolated from mice and cut into small pieces. The fragments were digested in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1% collagenase A (Roche)/0.1% dispase (Roche), and 10 U/ml DNase I (Roche) at 37°C for 60 min. The lung fragments were filtrated through a 70‐µm Cell Strainer (Greiner Bio‐One) into phosphate‐buffered saline, containing 5 mM ethylenediaminetetraacetic acid and 0.5% FBS. The single cell suspensions were divided into 1 × 10⁶ cells each per analysis and underwent a flow cytometric analysis with a FACS Versa (BD Biosciences‐Pharmingen). For the staining, fluorescence‐conjugated monoclonal antibodies against the following targets were used at a concentration of 0.25 µg/10⁶ cells in 100 µl volume: CD45 APC/Cy7 or PerCP/Cy5.5, CD11b PECy7 or PerCP/Cy5.5, CD11c PECy7, Gr‐1 PECy7 or APC, CD34 PECy7, CD40 PECy7, CD64 PECy7, CD80 PECy7, CD86 PECy7, IA/IE PECy7, F4/80 PECy7 or APC, CXCR4 biotin, Sca‐1 PECy7, Ly6C PECy7, Ly6G PECy7, MerTK PECy7, all from BioLegend, as were 7‐AAD Viability Staining Solution and PECy7‐conjugated Streptavidin. Each analysis of flow cytometry was performed through three experiments with a total of six mice per group.

Kawano H., Koyama K., Nishimura H., Toyoda Y., Kagawa K., Sato S., Naito N., Goto H., Inagaki Y, & Nishioka Y. (2020). Development of improved method to identify and analyze lung fibrocytes with flow cytometry in a reporter mouse strain. Immunity, Inflammation and Disease, 9(1), 120-127.

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Isolation and Characterization of Lung Immune Cells

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After the lung tissues collected, they were cut into fine pieces and digested with 1× collagenase in DMEM media for 1 h. The cells were passed through a 100-µm filter, and red blood cells were lysed with lysis buffer. The single cells were stained with CD45-AF700, Gr-1-BV605, CD64-PECy7, SiglecF-BV421, CD11b-AF594, CD206-AF488 (BioLegend). The samples were run on Flow Fortessa (BD Biosciences) and data were analyzed by Kaluza Analysis 2.1 (Beckman). The cells were first gated for live single cells followed by gating strategies required for different cell type. For cell sorting, samples were run on Moflo Astrios and cells were collected in DMEM media until further processed. For biodistribution analysis, the different immune cells were gated following a published protocol (55 (link)).

Singh A., Chakraborty S., Wong S.W., Hefner N.A., Stuart A., Qadir A.S., Mukhopadhyay A., Bachmaier K., Shin J.W., Rehman J, & Malik A.B. (2022). Nanoparticle targeting of de novo profibrotic macrophages mitigates lung fibrosis. Proceedings of the National Academy of Sciences of the United States of America, 119(15), e2121098119.

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Intestinal Tissue-Resident Macrophage Analysis

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For flow cytometric analysis of intestinal TRMs in the lamina propria, small intestine segments were washed, flattened, cut into 1.5cm pieces and shaken in 2 changes of HBSS with 5% FBS and 2 mM EDTA for 20 min at 37C. The suspension was passed through a mesh strainer to remove epithelial cells. The intestinal pieces were chopped using scissors and shaken in digestion media containing HBSS (Gibco) with 5% FBS, 1mg/ml Collagenase D, 2U/ml DNase I, 0.1U/ml Dispase for 30 mins at 37C. The digested samples were then vortexed briefly and passed through a 100-micron filter followed by staining for flow cytometry.
Cells were stained with the following fluorochrome-conjugated surface antibodies: CD45-BUV395 (BD Biosciences; Clone 30-F11), CD64-PE/Cy7 (Biolegend; Clone X54-5/7.1), CD11c-Alexa Fluor 488 (eBioscience; Clone N418), CD11b-APC/Cy7 (Biolegend; Clone M1/70), CX3CR1-BV785 (Biolegend; Clone SA011F11), and F4/80-APC (eBioscience; Clone BM8). Cellular fluorescence was measured using an LSRII Fortessa flow cytometer, and data were analyzed using FlowJo software (Tree Star).

Martin A.T., Giri S., Safronova A., Eliseeva S.I., Kwok S.F, & Yarovinsky F. (2024). Parasite-induced IFN-γ regulates host defense via CD115 and mTOR-dependent mechanism of tissue-resident macrophage death. PLOS Pathogens, 20(2), e1011502.

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Multiparametric Flow Cytometry Analysis

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Cell suspensions were incubated with Ghost Dye BV510 Live/Dead stain (Tonbo Biosciences, San Diego, CA) at room temperature for 20 min, washed, incubated with 1:250 Fc block (2.4G2, BD Biosciences, Franklin Lakes, NJ) at 4 °C for 10 min, and then incubated with fluorochrome-labeled antibodies at 4C for 30 min using the following antibodies: CD19-PerCP/Cyanine5.5 (1:400; 6D5, Biolegend), CD64-PE/Cy7 (1:200; X54-5/7.1, Biolegend), Ly6G-PE (1:200; 1A8, Biolegend), CD11b-APC-780 (1:200; M1/70, eBioscience), CD45-AF700 (1:200; 30-F11, eBioscience), F4/80-APC (1:100; BM8, Biolegend), CD90.2-BV570 (1:100; 30-H12, Biolegend), Gr-1-BV711 (1:200; RB6-8C5, Biolegend) , CD11c-Bv650 (1:200; N418, Biolegend), Ly6c-Bv605 (1:100; HK1.4, Biolegend), MHC Class II-e450 (1:400; M5/114.15.2, eBioscience). Cells were analyzed using an LSRII flow cytometer (BD Biosciences) through the UCSF Liver Center Flow Cytometry Core Facility. Analysis was performed with FlowJo v.10.7.1 (Tree Star, Ashland, OR).

Lopez-Ichikawa M., Vu N.K., Nijagal A., Rubinsky B, & Chang T.T. (2021). Neutrophils are important for the development of pro-reparative macrophages after irreversible electroporation of the liver in mice. Scientific Reports, 11, 14986.

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Comprehensive Flow Cytometry Panel

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The following Abs were used for flow cytometry: CD115 Alexa Fluor 488 (AFS98, BioLegend, 135512), CD93 PerCP-Cy5.5 (AA4.1, BioLegend, 136512), CD144 PE (11D4.1, BD Biosciences, 562243), F4/80 PE-CF594 (T45-2342, BD Biosciences, 565613), CD64 PE-Cy7 (X54-5/7.1, BioLegend, 139314), Sca-1 allophycocyanin (D7, BioLegend, 108112), CD16/32 allophycocyanin-R700 (2.4G2, BD Biosciences, 565502), CD11c allophycocyanin-780 (N418, eBio-science, 47011482), CD41 BV421 (MWReg30, BD Biosciences, 747729), Ter119 BV510 (TER-119, BD Biosciences, 563995), Ly6G BV605 (1A8, BD Biosciences, 563005), CX3CR1 BV650 (SA011F11, BioLegend, 149033), c-Kit BV711 (2B8, BD Biosciences, 105835), CD45 BV786 (30-F11, BD Biosciences, 564225), CD3e BUV395 (145-2C11, BD Biosciences, 563565), CD11b BUV737 (M1/70, BD Biosciences, 564443), CD68 PerCP-Cy5.5 (FA-11, BioLegend, 137010), VCAM1 Alexa Fluor 647 (429, BioLegend, 105712), MHC class II Alexa Fluor 700 (M5/114, Thermo Fisher Scientific, 56532182), Siglec-F BV421 (E50-2440, BD Biosciences, 562681), Tim-4 BV510 (21H12, BD Biosciences, 742774), Live/Dead, fixable UV (Thermo Fisher Scientific, L34962).

Philpott J., Kazimierczyk S., Korgaonkar P., Bordt E., Zois J., Vasudevan C., Meng D., Bhatia I., Lu N., Jimena B., Porter C., Cherayil B.J, & Jain N. (2022). RXRα Regulates the Development of Resident Tissue Macrophages. ImmunoHorizons, 6(6), 366-372.

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Comprehensive Flow Cytometry Panel

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Multicolor Flow Cytometry of Immune Cells

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Flow cytometry data were acquired using a Cytek Aurora equipped with violet (405 nm), blue (488 nm), and red (640 nm) lasers. The following antibodies and stains were used: CCR2-BV421 (clone 475301, 747963; BD), Ly6C-BV510 (clone HK1.4, 128033; Biolegend), B220-BV605 (clone RA3-6B2, 103243; Biolegend), Ly6G-BV711 (clone 1A8, 127643; Biolegend), CD11c-BV785 (clone N418, 117336; Biolegend), CX3CR1-A488 (clone SA011F11, 149021; Biolegend), CX3CR1-BV785 (clone SA011F11, 149029; Biolegend), CD163-PE (clone TNKUPJ, 12-1631-80; Thermo Fisher Scientific), CD64-PE/Cy7 (clone X54-5/7.1, 139313; Biolegend), MRC1-PCP5.5 (clone C068C2, 141716; Biolegend), CD11b-PEFire640 (clone M1/70, 101279; Biolegend) TCRb-APC (clone H57-597, 109212; Biolegend), MHCII-A700 (clone M5/114.15.2, 107622; Biolegend), XCR1-APC/Cy7 (clone ZET, 148223; Biolegend), CD45-APC-Fire810 (clone 30F11, 103173; Biolegend), CD45.1-A647 (clone A20, 110720; Biolegend), CD45.2- BV785 (clone 104, 109839; Biolegend), and LIVE/DEAD Violet dead cell stain kit (L34955; Thermo Fisher Scientific). Flow cytometry data was analyzed using Flowjo 10 software. Dimensionality reduction and cluster identification were performed using the UMAP and Phenograph packages, respectively.

Lund H., Hunt M.A., Kurtović Z., Sandor K., Kägy P.B., Fereydouni N., Julien A., Göritz C., Vazquez-Liebanas E., Andaloussi Mäe M., Jurczak A., Han J., Zhu K., Harris R.A., Lampa J., Graversen J.H., Etzerodt A., Haglund L., Yaksh T.L, & Svensson C.I. (2023). CD163+ macrophages monitor enhanced permeability at the blood–dorsal root ganglion barrier. The Journal of Experimental Medicine, 221(2), e20230675.

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