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  • br In this paper we describe two in


    In this paper, we describe two in vitro phagocytosis methods that utilize fluorescent dyes to assess cancer cell engulfment by macro-phages, which can easily be applied to other phagocyte processes (Fig. 1). The first is the flow cytometry–based phagocytosis assay, which is a simple and effective method for analyzing the phagocytic activity of macrophages using carboxyfluorescein succinimidyl ester (CFSE) or CellTracker dye (Willingham et al., 2012). CellTracker dye,
    Abbreviations: BMDMs, bone-marrow derived macrophages; DCs, dendritic cells; APCs, antigen presenting cells; pHrodo-SE, pHrodo-succinimidyl ester; M-CSF, macrophage colony-stimulating factor; CMFDA, 5-Chloromethylfluorescein diacetate Corresponding authors at: Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.
    E-mail addresses: [email protected] (Y. Yang), [email protected] (I.-S. Kim). 1 These authors contributed equally to this work.
    Fig. 1. The schematic method of cancer phagocytosis assay utilizing CellTracker or pHrodo-SE fluorescence dyes.
    which can penetrate cell membranes, yields higher fluorescent signals at physiological pH. The degree of phagocytosis can be confirmed by tracing the co-localization of fluorescence detected from cancer Leupeptin and macrophages stained with distinguishable CellTracker dyes.
    However, the flow cytometry–based phagocytosis assay provides limited information and may overestimate the actual potential of the phagocytosis of cancer cells as a result of the inappropriate detection of intercellular binding. Therefore, we suggest an optimized protocol to determine the phagocytosis of cancer cells based on fluorescence mi-croscopy using pHrodo-succinimidyl ester (pHrodo-SE) dye. The pHrodo-SE dye utilized for the labeling of cancer cells reacts with the primary amines on cancer cells to yield covalently linked pH probes, which display increased fluorescence as the environmental pH becomes more acidic (Miksa et al., 2009). Due to the low pH of the phagolyso-some, engulfed cancer cells can be visualized by pHrodo-SE staining, which can be distinguished from cancer cells merely adhering to the outer cell surface of the phagocytes. Hence this technique allows for the straightforward quantification of phagocytosis of cancer cells using fluorescence microscopy.
    These phagocytosis assays can be applied to multiple types of tu-mors or phagocytes and be used to evaluate the efficacy of various phagocytosis enhancers. A better understanding of the interactions between tumor cells and phagocytes through in vitro phagocytosis as-says may contribute to the identification of novel targets for cancer immunotherapy. 
    2. Materials and methods
    2.1. Reagents and animals
    Recombinant murine macrophage colony–stimulating factor (M-CSF) was purchased from Peprotech. CellTracker 5-Chloromethylfluorescein diacetate (CMFDA), CellTracker™ Deep Red, pHrodo™ Red SE, and β-mercaptoethanol were purchased from Thermo Fisher Scientific Inc. APC-anti-F4/80 (clone BM8, 123116, 1:100) and APC-rat IgG2a, ĸ Isotype Ctrl antibody (clone RTK2758, 400512, 1:100) were obtained from BioLegend.
    Male BALB/c white mice (8 weeks old) were purchased from Orient Bio Inc. All mice were bred and maintained under specific pathogen-free conditions at the Korea Institute of Science and Technology (KIST). The study protocols were performed following guidelines of the Institutional Animal Care and Use Committee (IACUC) of the KIST.
    Murine 4 T1-Luc breast cancer cells, murine CT26.CL25 colon ade-nocarcinoma, and human HT29 colon adenocarcinoma were cultured in RPMI-1640 (Hyclone) containing 10% fetal bovine serum (Atlas) and 1% antibiotic-antimycotic (Thermo). Murine B16F10-Ova melanoma cells were cultured in DMEM (Hyclone) containing 10% fetal bovine serum (Atlas) and 1% antibiotic-antimycotic (Thermo). All cell lines were maintained in 5% CO2 at 37 °C.
    To generate bone marrow-derived macrophages (BMDMs), bone marrow cells were isolated from the leg bones of BALB/c mice. Isolated bone marrow cells were grown in RPMI-1640 medium (Welgene) sup-plemented with 10% fetal bovine serum (Gibco) and 1% antibiotic-
    Fig. 2. Phagocytosis assay using CellTracker dye and pHrodo-SE. (a) Schematic diagram of experiments for differentiation of BMDMs. (b) Representative histograms showing the differentiation of BMDMs with anti-F4/80 antibody for phagocytosis assays. (c) Left: Representative FACS plots showing the phagocytosis of a range of 4 T1-Luc cells stained with CellTracker by BMDMs. Right: Phagocytosis was calculated as the percentage of double-positive BMDMs among CellTracker Deep Red-positive BMDMs, assessed by FACS analysis. Data are presented as means ± S.D. (n = 5–6). (d) Left: Representative microscopic images of pHrodo-SE phagocytosis assays performed using CMFDA-stained BMDMs (green) against 4 T1-Luc, B16F10-Ova or CT26.CL25 cells labeled with pHrodo-SE. Scale bars: 100 μm. Right: Phagocytosis was calculated from fluorescence microscopic images. Data are presented as means ± S.D. (n = 6–9). The phagocytosis percentage indicates the percentage of BMDMs containing cancer cells per total BMDMs. ***p < .001 by Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)