F4 80 percp cy5

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F4/80-PerCP/Cy5.5 is a fluorochrome-conjugated antibody that recognizes the F4/80 antigen, which is expressed on the surface of macrophages. This antibody can be used in flow cytometry applications to identify and quantify macrophage populations in various biological samples.

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F4/80 (349 citations) PerCP-Cy5.5 (133 citations) PerCP/Cy5.5 anti-mouse F4/80 (4 citations) Anti-F4/80-PerCP/Cy5.5 (3 citations) PerCP/Cy5.5-F4/80 (clone BM8), (2 citations) PerCP‐cy5.5‐conjugated anti‐F4/80 (2 citations) PerCP/Cy5.5-conjugated anti-mouse F4/80 antibody (2 citations) PerCP-Cy5.5-anti-F4/80 (2 citations)

14 protocols using f4 80 percp cy5

Flow Cytometric Analysis of Immune Cells

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For flow cytometric analysis, cells were blocked with PBS containing 1% bovine serum albumin and 0.1% rat IgG (Sigma-Aldrich, St. Louis, USA) for 30 min. After a washing step, cells were stained with SiglecF-PE or -AlexaFluor647 (Clone: E50-2440, BD Pharmingen, San Diego, USA), F4/80-PerCP-Cy5.5 (Clone: BM8), Ly6G-PE (Clone: 1A8), Ly6C-APC-Cy7 (Clone HK1.4) (all BioLegend, San Diego, USA), and CD11b-FITC or -PE-Cy7 (Clone: M1/7; eBioscience, San Diego, USA). For intracellular staining, cells were incubated with fixation and permeabilization buffer (eBioscience) overnight. Cells were stained with rabbit anti-mouse RELMα (Peprotech, Rocky Hill, USA) followed by donkey anti-rabbit IgG AlexaFluor647 (Clone: Poly4064, BioLegend) and CD86-PE (Clone: GL1) and MHCII-APC (Clone: M5/114.15.2, eBioscience) to determine cell activation. The gating strategy used to identify macrophages, monocytes, eosinophils and neutrophils is shown in Figure 1 (Fig. 1).
IFNγ , IL-10, TGFβ and TNF (all eBioscience) as well as CXCL1/KC and CXCL2/MIP-2 (both R&D, Minneapolis, USA) were measured from peritoneal lavage and serum by ELISA according to the manufacturers’ protocols and analyzed using a plate reader (Molecular Devices) with SoftMax Pro 6.

Buerfent B.C., Ajendra J., Stamminger W., Gondorf F., Hoerauf A, & Hübner M.P. (2019). TGFβ depletion does neither modulate acute E. coli-induced inflammatory immune responses nor impair the protective effect by chronic filarial infection. GMS Infectious Diseases, 7, Doc04.

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Immune Cell Profiling of Islets

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Islets were isolated from ZBTB46-DTR BM chimera mice dissociated as previously described. Islet samples were stained with the fluorescent-labeled antibodies CD45 BUV395 (BD), CD90.2 PE-Cy7 (BioLegend), CD19 PE (BioLegend), F4/80 PerCP-Cy5.5 (BioLegend), MHC-II FITC (BioLegend), CD11c BUV805 (BD), CD11b APC-Cy7 (BioLegend), XCR1 BV785 (BioLegend), unlabeled goat α-MERTK (R&D Systems), and donkey α-goat secondary (AF647; Jackson ImmunoResearch).

Lindsay R.S., Whitesell J.C., Dew K.E., Rodriguez E., Sandor A.M., Tracy D., Yannacone S.F., Basta B.N., Jacobelli J, & Friedman R.S. (2021). MERTK on mononuclear phagocytes regulates T cell antigen recognition at autoimmune and tumor sites. The Journal of Experimental Medicine, 218(10), e20200464.

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Multiparametric Flow Cytometry Analysis

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Flow-cytometry antibodies included CD3e-PE, PD1-BV605, CD45-BV786 (BD-Biosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-APC, CD45.1-PE (eBioscience), CD45.2-Alexa700, CD-8α-Percp-Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas-red (Life Technologies, Carlsbad, CA, USA). Blocking PD-1 antibody (clone RMP1-14, BioXCell, Branford, CT, USA) was administered systemically by intraperitoneal injections of 250 μg [41 (link)]. Blocking anti-CD40L (clone MR1, BioXCell) was administered systemically by intraperitoneal injections of 250 μg [19 (link)].

Sharon S., Baird J.R., Bambina S., Kramer G., Blair T.C., Jensen S.M., Leidner R.S., Bell R.B., Casap N., Crittenden M.R, & Gough M.J. (2021). A platform for locoregional T-cell immunotherapy to control HNSCC recurrence following tumor resection. Oncotarget, 12(13), 1201-1213.

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Quantification of Immune Cell Subsets

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GO dispersion was purchased from Xianfeng Nano Technology Co., Ltd, Nanjing, China. Squalene was from Macklin Biochemical Co., Ltd, Shanghai, China. Pgp3 was purified by affinity resin with the glutathione S-transferases (GST) tag removed. ELISA MAX™ Deluxe mouse kits were purchased from Biolegend USA. Fluorescein isothiocyanate (FITC) goat anti-mouse IgG was purchased from Absin Bioscience Inc and Cy7-BSA was purchased from Xian Qiyue Biology. Fluorescently labelled antibody CD45-PE, CD11b-APC, CD11b-PerCP Cy5.5, PE-CD86, CD206-FITC, F4/80-FITC, and F4/80-PerCP Cy5.5 were purchased from Biolegend. Specific pathogen-free BALB/c mice (SYXK-2020-0002) were bought from SJA Laboratory Animal Co., Ltd, Hunan, China. All animal experiments were carried out with the authorization of the animal ethics committee of the University of South China. All procedures were performed in accordance with the Laboratory Animal Management Regulations in China.

Zhao L., Shu M., Chen H., Shi K, & Li Z. (2023). Preparation of graphene oxide–stabilized Pickering emulsion adjuvant for Pgp3 recombinant vaccine and enhanced immunoprotection against Chlamydia Trachomatis infection. Frontiers in Immunology, 14, 1148253.

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Liver Cell Isolation and Characterization

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After digestion of the whole liver with pronase and collagenase, cells were centrifuged and incubated with CD16/32 antibodies (16–0161, eBioscience) to block Fc receptors. Then, cells were incubated with antibodies against CD45-APC-Cy7 (557659, BD Pharmingen), Ly-6G-PE (551461), CD11b-APC (553312), Ly-6C-FITC (553104), and/or F4/80-PerCP/Cy5.5 (123128, BioLegend) at 50-fold dilution for 30 min on ice. After washing, the cells were incubated with DAPI for detection of dead cells. We compensated each fluorescence signal using cells with or without staining in each fluorochrome with FACS Aria I (BD Bioscience) in the USC Flow Cytometry Core.
Other method details are provided in the Supporting Information.

Balog S., Li Y., Ogawa T., Miki T., Saito T., French S.W, & Asahina K. (2019). Development of Capsular Fibrosis beneath the Liver Surface in Humans and Mice. Hepatology (Baltimore, Md.), 71(1), 291-305.

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Multiparameter Flow Cytometry Analysis

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Thoracic cavity, spleen, BAL and skin cells were analyzed by flow cytometry. Cells were isolated as described above and subsequently blocked with PBS/1% BSA including 0.1% rat IgG (Sigma) and stained. Flow cytometric analysis was performed using a combination of the following surface markers: CD4-FITC, CD8-APC, SiglecF-PE, F4/80-PerCP-Cy5.5, CD11b-APC-Cy7 (all BioLegend), and Ly6G-PE-Cy7 (ThermoFisher). Thoracic cavity and spleen CD4⁺ T cells and CD8⁺ T cells were identified as CD4^high or CD8^high cells, respectively; neutrophils as Ly6G^high, CD4^low; eosinophils as SiglecF^high, F4/80^low; macrophage populations were identified as F4/80^high, SiglecF^low. BAL macrophages were identified as F4/80^high, SiglecF^high and neutrophils as Ly6G^high/CD11b^high. Cells were further analyzed with MHCII-PE (BioLegend) in a different panel. Cells were stained and after another wash and centrifugation step cells were analysed by flow cytometry. Analysis was performed using a BD FACS Canto system and data was subsequently analyzed using the FACS Diva 5.1 software (BD Biosciences). During analysis, cut-offs were set using the fluorescence minus one (FMO) approach. The gating strategy is shown in S1 Fig.

Frohberger S.J., Fercoq F., Neumann A.L., Surendar J., Stamminger W., Ehrens A., Karunakaran I., Remion E., Vogl T., Hoerauf A., Martin C, & Hübner M.P. (2020). S100A8/S100A9 deficiency increases neutrophil activation and protective immune responses against invading infective L3 larvae of the filarial nematode Litomosoides sigmodontis. PLoS Neglected Tropical Diseases, 14(2), e0008119.

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Multi-Parameter Immune Cell Analysis

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Antibodies against murine antigens (BioLegend, Mexico-City): APC-anti-L-selectin, Cy7-anti-CD45, ICAM-1 (clone YN1.1, uncoupled) with secondary AF488-goat-anti-mouse, PE-anti-CD117 (clone ACK2), APC-anti-Ly6G (clone 1A8), F4/80-PerCP/Cy5.5, CD3-PE, CD19-BV421, CD45-FITC, APC-anti-CD106 (clone 429). Alexa647-anti-HS1 (clone D5A9 XP) was purchased from Cell Signaling Technology.

Guerrero-Fonseca I.M., García-Ponce A., Vadillo E., Lartey N.L., Vargas-Robles H., Chánez-Paredes S., Castellanos-Martínez R., Nava P., Betanzos A., Neumann B.M., Penkala-Auguste K., Lefort C.T, & Schnoor M. (2022). HS1 deficiency protects against sepsis by attenuating neutrophil-inflicted lung damage. European journal of cell biology, 101(2), 151214.

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Multi-Parameter Immune Cell Analysis

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Comprehensive Immune Cell Profiling

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We used the following gating schemes: total leukocytes (CD45.2⁺), total B cells (CD19⁺ CD3⁻), B-2 (CD19⁺ CD3⁻ B220^hi CD5⁻ CD23⁺), B-1a (CD19⁺ CD3⁻ IgM⁺ IgD⁻ B220^lo CD5⁺ CD23⁻), B-1b (CD19⁺ CD3⁻ IgM⁺ IgD⁻ B220^lo CD5⁻ CD23⁻), regulatory B [Breg] cells (CD19⁺ B220⁺ CD22⁺ CD5⁻ IgM⁺ IgD⁺), total T cells (CD19⁻ CD3⁺), Treg cells (CD19⁻ CD3⁺ CD4⁺ CD25⁺), and macrophages (CD19⁻ CD3⁻ CD11b⁺ F4/80⁺). Dead cells were distinguished by Live/Dead Fixable Aqua staining (Life Technologies). Baselines for IL-10 EGFP mice were set using age- and diet-matched C57BL/6J mice. For macrophage intracellular cytokine staining, cells were stimulated with LPS (1 μg/mL; Sigma-Aldrich) and brefeldin A (5 μg/mL; BioLegend) overnight and stained using the Cytofix/Cytoperm Kit (BD Biosciences) according to the vendors’ instructions. Data were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).
CD16/32, CD3-PacBlue, CD5-PE-Cy5, CD19-PerCP-Cy5.5, CD22-PE, CD23-PE-Cy7, CD25-PE, CD45.2-APC, B220-APC-Cy7, F4/80-PE, F4/80-PErCP-Cy5.5, IgD-PE, and TNF-α–PE antibodies were from BioLegend. IgM-efluor650, CD4-efluor650, and CD11b-efluor605 antibodies were from eBioscience.

Shen L., Chng M., Alonso M.N., Yuan R., Winer D.A, & Engleman E.G. (2014). B-1a Lymphocytes Attenuate Insulin Resistance. Diabetes, 64(2), 593-603.

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Comprehensive Murine and Human Immune Profiling

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Flow cytometry antibodies for murine samples included CD3e-PE, PD1-BV605, CD45-BV786 (BDbiosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-PE (ebioscience), CD45.2-Alexa700, CD-8α-Percp Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas red (life technologies), CD278 (ICOS)-PE (Biolegend). Flow cytometry antibodies for human samples included CD45-BV510, CD3-Alexa700, CD4-BV785, CD8-BV711, CTLA-4-PE/Dazzle 594, 4-1BB-PE (Biolegend), CD39-BV650, PD-1-PE-Cy7 and Ki-67 Alexa 488 (BD Biosciences), CD103-APC and FOXP3-Alexa 700 (eBioscience).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).

Sharon S., Duhen T., Bambina S., Baird J., Leidner R., Bell B., Casap N., Crittenden M., Vasudevan S., Jubran M., Kravchenko-Balasha N, & Gough M. (2021). Explant Modeling of the Immune Environment of Head and Neck Cancer. Frontiers in Oncology, 11, 611365.

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