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This work was supported by the Cancer Biology and Precision Therapeutics Center, China Medical University, from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (CBPTC-PROJ-5-2).
We thank the Cancer Biology and Precision Therapeutics Center for providing funding and academic support.

<ol>
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U., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sites+of+distant+recurrence+and+clinical+outcomes+in+patients+with+metastatic+triple-negative+breast+cancer:+High+incidence+of+central+nervous+system+metastases.">Sites of distant recurrence and clinical outcomes in patients with metastatic triple-negative breast cancer: High incidence of central nervous system metastases.</a> Cancer. 113 (10), 2638-2645 (2008).</li><li>Bianchini, G., De Angelis, C., Licata, L., Gianni, L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+landscape+of+triple-negative+breast+cancer+-+expanded+options,+evolving+needs.">Treatment landscape of triple-negative breast cancer - expanded options, evolving needs.</a> Nat Rev Clin Oncol. 19 (2), 91-113 (2022).</li><li>Lecoultre, M., Dutoit, V., Walker, P. R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+function+of+tumor-associated+macrophages+as+a+key+determinant+of+tumor+progression+control:+A+review.">Phagocytic function of tumor-associated macrophages as a key determinant of tumor progression control: A review.</a> J Immunother Cancer. 8 (2), (2020).</li><li>Mantovani, A., Allavena, P., Marchesi, F., Garlanda, C. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+as+tools+and+targets+in+cancer+therapy.">Macrophages as tools and targets in cancer therapy.</a> Nat Rev Drug Discov. 21 (11), 799-820 (2022).</li><li>Chan, S. H., Lin, C. Y., Tseng, H. J., Wang, L. H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cd24a+knockout+results+in+an+enhanced+macrophage-+and+cd8&#x207A;+t+cell-mediated+anti-tumor+immune+responses+in+tumor+microenvironment+in+a+murine+triple-negative+breast+cancer+model.">Cd24a knockout results in an enhanced macrophage- and cd8&#x207A; t cell-mediated anti-tumor immune responses in tumor microenvironment in a murine triple-negative breast cancer model.</a> J Biomed Sci. 32 (1), 73 (2025).</li><li>Majeti, R., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD47+is+an+adverse+prognostic+factor+and+therapeutic+antibody+target+on+human+acute+myeloid+leukemia+stem+cells.">CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells.</a> Cell. 138 (2), 286-299 (2009).</li><li>Barkal, A. A., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD24+signalling+through+macrophage+siglec-10+is+a+target+for+cancer+immunotherapy.">CD24 signalling through macrophage siglec-10 is a target for cancer immunotherapy.</a> Nature. 572 (7769), 392-396 (2019).</li><li>Wang, X., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD24-siglec+axis+is+an+innate+immune+checkpoint+against+metaflammation+and+metabolic+disorder.">CD24-siglec axis is an innate immune checkpoint against metaflammation and metabolic disorder.</a> Cell Metab. 34 (8), 1088-1103.e6 (2022).</li><li>Chan, S. H., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+novel+role+of+CD24+as+an+oncogenesis+regulator+and+therapeutic+target+for+triple-negative+breast+cancer.">Identification of the novel role of CD24 as an oncogenesis regulator and therapeutic target for triple-negative breast cancer.</a> Mol Cancer Ther. 18 (1), 147-161 (2019).</li><li>Huang, S., Zhang, X., Wei, Y., Xiao, Y. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Checkpoint+CD24+function+on+tumor+and+immunotherapy.">Checkpoint CD24 function on tumor and immunotherapy.</a> Front Immunol. 15, 1367959 (2024).</li><li>Weiskopf, K., Weissman, I. L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+are+critical+effectors+of+antibody+therapies+for+cancer.">Macrophages are critical effectors of antibody therapies for cancer.</a> MAbs. 7 (2), 303-310 (2015).</li><li>Cao, X., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoting+antibody-dependent+cellular+phagocytosis+for+effective+macrophage-based+cancer+immunotherapy.">Promoting antibody-dependent cellular phagocytosis for effective macrophage-based cancer immunotherapy.</a> Sci Adv. 8 (11), eabl9171 (2022).</li><li>Aderem, A., Underhill, D. M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+phagocytosis+in+macrophages.">Mechanisms of phagocytosis in macrophages.</a> Annu Rev Immunol. 17, 593-623 (1999).</li><li>Flannagan, R. S., Jaumouille, V., Grinstein, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+phagocytosis.">The cell biology of phagocytosis.</a> Annu Rev Pathol. 7, 61-98 (2012).</li><li>Denardo, D. G., Ruffell, B. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+as+regulators+of+tumour+immunity+and+immunotherapy.">Macrophages as regulators of tumour immunity and immunotherapy.</a> Nat Rev Immunol. 19 (6), 369-382 (2019).</li><li>Horsthemke, M., Wilden, J., Bachg, A. C., Hanley, P. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+3d+imaging+of+phagocytosis+by+mouse+macrophages.">Time-lapse 3d imaging of phagocytosis by mouse macrophages.</a> J Vis Exp. (140), e57566 (2018).</li></ol>

The authors disclose no conflict of interest.

Several steps in this protocol are critical for obtaining reliable and reproducible results. First, the quality and purity of PBMC isolation directly influence macrophage yield and differentiation efficiency. Careful layering of diluted blood over the Ficoll gradient and centrifugation with the brake turned off are essential to preserve the buffy coat interface. Incomplete RBC lysis or excessive incubation with lysis buffer will compromise PBMC viability. Second, proinflammatory macrophage polarization must be verified b...

Breast cancer is the most frequently diagnosed malignancy among women worldwide, accounting for approximately 2.3 million new cases annually1. The disease is clinically heterogeneous and is classified into at least four major molecular subtypes based on the status of hormone receptors—estrogen receptor (ER) and progesterone receptor (PR)—and the expression of the human epidermal growth factor receptor 2 (HER2) oncogene2. Luminal A tumors express ER and/or PR without HER2 overexpression (ER/PR+ and HER2−); luminal B tumors co-express ER/PR and HER2 (ER/PR+ and HER2+); HER2+ tumors are characterized by high HER2 expression independent of hormone receptor status; and triple-negative breast cancer (TNBC) lacks expression of all three markers (ER−, PR−, and HER2−)2,3. Among these subtypes, TNBC represents the most clinically challenging form and accounts for approximately 15%–20% of all breast cancers. Patients with TNBC exhibit the most aggressive disease course, with the worst prognosis and shortest 5-year overall survival, owing to poor responses to conventional chemotherapy and the absence of effective targeted therapies3,4. Furthermore, TNBC displays a significantly high propensity for visceral metastasis, particularly to the lungs within 5 years of diagnosis, and patients with advanced or metastatic TNBC (mTNBC) have a median survival that rarely exceeds 2 years5,6. These statistics underscore the critical and unmet need for novel therapeutic strategies for TNBC.
The tumor microenvironment (TME) plays a pivotal role in breast cancer progression and immune evasion. Tumor-associated macrophages rank among the most abundant immune cells within the TME and have recently been recognized as critical mediators of innate anti-tumor immunity7,8,9. While TAMs were historically divided into pro-inflammatory “M1” and pro-tumoral “M2” states, this binary model fails to capture the true extent of macrophage heterogeneity. Instead, TAMs occupy a dynamic functional spectrum, evolving in response to the diverse biochemical signals within the TME. Within this complex landscape, cancer cells actively subvert macrophage activity through various immune evasion strategies, notably the overexpression of “don’t eat me” signals. The CD47/SIRPα axis was the first identified innate immune checkpoint in this context6,10, and more recently, CD24 has emerged as a novel and potent antiphagocytic signal11,12. CD24 is a small and heavily glycosylated mucin-type glycoprotein anchored to the cell membrane via a glycosylphosphatidylinositol linkage13. It is expressed on various immune cell lineages under physiological conditions but is frequently overexpressed in solid tumors, including breast, ovarian, pancreatic, and colorectal cancers14. On tumor cells, CD24 engages the inhibitory receptor sialic acid–binding immunoglobulin-like lectin 10 (Siglec-10) expressed on macrophages. This interaction triggers phosphorylation of the immunoreceptor tyrosine-based inhibitory motif domains in the cytoplasmic tail of Siglec-10, activating the SHP-1 and SHP-2 phosphatases that suppress downstream phagocytic signaling11. Blockade of the CD24/Siglec-10 interaction with monoclonal antibody has been shown to robustly augment the phagocytosis of CD24-expressing human tumors by macrophages. Our recent study also showed that knockout of CD24a in breast cancer cells increases their susceptibility to macrophage phagocytosis in a murine triple-negative breast cancer model9.
Antibody-dependent cellular phagocytosis (ADCP) is a pivotal mechanism by which antibodies direct macrophages to phagocytose tumor cells15,16. Upon binding to tumor-expressed antigens, the Fc region of the antibody engages Fcγ receptors (FcγRs) on macrophages, triggering intracellular signaling cascades that culminate in cytoskeletal rearrangement, phagocytic cup formation, and engulfment of the target cell17.
In this study, we developed and validated a time-lapse live-cell imaging protocol for measuring antibody-driven macrophage phagocytosis of breast cancer cells. The method utilizes peripheral blood mononuclear cell (PBMC)-derived macrophages from healthy human donors, differentiated with macrophage colony-stimulating factor (M-CSF) and subsequently stimulated with lipopolysaccharide (LPS) and interferon gamma (IFN-γ). Flow cytometry analysis confirmed upregulation of CD80 within the CD11b⁺ population following stimulation (Figure 1), supporting the classically activated phenotype. This activation state confers robust antibody-mediated phagocytic capacity, making it well suited for evaluating antibody-dependent cellular phagocytosis (ADCP). GFP-tagged CD24-positive breast cancer cells serve as target cells and are opsonized with a therapeutic anti-CD24 monoclonal antibody prior to co-culture. Phagocytic events are recorded over a 16 h period using an automated microscope under controlled environmental conditions. To enable quantitative assessment in parallel with dynamic visualization, a separate endpoint assay is performed under identical co-culture conditions, allowing rigorous measurement of phagocytosis following removal of non-internalized target cells. This protocol provides a standardized, reproducible, and visually informative platform for evaluating ADCP mediated by an anti-CD24 antibody. Importantly, the integration of time-lapse imaging with a complementary endpoint-based quantification strategy enables simultaneous evaluation of phagocytic kinetics and magnitude. The method can be adapted for investigating other macrophage-directed immunotherapeutic targets. This protocol is most suitable for evaluating ADCP mediated by therapeutic monoclonal antibodies against surface antigens on cancer cells. It is well suited for comparative screening of candidate antibodies, dose-response characterization, and mechanistic studies of Fc–FcγR engagement, including the effects of “don’t-eat-me” signal blockade (e.g., CD47–SIRPα). Because the assay uses primary human macrophages, it is also appropriate for translational studies that require assessment of donor-to-donor variability prior to clinical development.
<img alt="Macrophage differentiation flowchart; PBMCs isolation, centrifuge, flow cytometry result, CD markers." class="xfigimg" src="/files/ftp_upload/71461/71461fig1.jpg" data-altupdatedat="1790654420000" data-alt="Figure 1"> 
Figure 1. Generation and flow cytometry validation of IFN-γ/LPS-stimulated macrophages derived from peripheral blood mononuclear cells. Upper panel: Peripheral blood mononuclear cells (PBMCs) are isolated from healthy donors by density gradient centrifugation using Ficoll solution at 400 × g for 30 min at 20°C, with all reagents pre-equilibrated to 20°C ± 2°C. Isolated PBMCs are cultured in ImmunoCult-SF medium supplemented with 100 ng/mL macrophage colony-stimulating factor (M-CSF) for 7 days to drive macrophage differentiation (Day 0–7), with medium supplementation at Day 5. On Day 8, differentiated macrophages are stimulated with 100 ng/mL IFN-γ and 1 µg/mL LPS for 48 h (Day 8–10). Cells are then subjected to flow cytometry analysis. Bottom panel: Flow cytometry analysis of macrophages. Cells were stained with a surface panel consisting of CD11b-FITC, CD80-PE, and CD206-PerCP-Cy5.5, followed by DAPI addition prior to acquisition for dead cell exclusion. Gating strategy: Cells were first gated on live cells (DAPI⁻), followed by CD11b⁺ identification on a CD11b-FITC versus FSC-A plot to confirm macrophage identity. Live CD11b⁺ events were then analyzed on a CD80 versus CD206 dot plot to confirm the CD80⁺CD206⁻ population induced by IFN-γ/LPS stimulation. Right: Representative dot plot of CD11b-FITC versus FSC-A showing that 99.8% of gated cells were CD11b⁺, confirming macrophage identity. Left: The majority of CD11b⁺ cells fell within the CD80⁺CD206⁻ gate (Q1: 95.2%), indicating the expected pro-inflammatory phenotype under the applied stimulation conditions. <a href="https://www.jove.com/files/ftp_upload/71461/71461fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Users should consider several constraints when applying this technique. First, donor variability in PBMC-derived macrophages can introduce substantial variance in phagocytic output, and we recommend using multiple donors (n ≥ 3) with individual donor data reported alongside pooled results. Second, the 2D co-culture format does not recapitulate the tumor microenvironment, and findings should be validated in 3D or in vivo models before clinical inference. Third, the 16-hour imaging window captures early phagocytic events but does not reflect longer-term processes such as antigen presentation or secondary immune activation. Thus, the combined use of real-time imaging and endpoint quantification provides a complementary framework that enhances both interpretability and reproducibility of ADCP measurements.

<table><tbody><tr><td>0.5 M Ethylenediaminetetraacetic acid (EDTA) stock solution</td><td>Sigma-Aldrich (or equivalent)</td><td>E7889 (or equivalent)</td><td>Used to prepare 10 mM EDTA working solution in PBS for cell detachment</td></tr><tr><td>24-well tissue culture plates (TC-treated)</td><td>Corning (or equivalent)</td><td>3524</td><td>Used for co-culture of macrophages and cancer cells during phagocytosis assay</td></tr><tr><td>Anti-human CD206 antibody (flow cytometry)</td><td>BioLegend (or equivalent)</td><td>321122 (or equivalent)</td><td>Marker for alternative macrophage phenotype</td></tr><tr><td>Anti-human CD24 monoclonal antibody</td><td>Thermo Fisher Scientific (or equivalent)</td><td>MA1-91384 (or equivalent)</td><td>Used to opsonize cancer cells for antibody-dependent phagocytosis</td></tr><tr><td>Automated live-cell imaging system with environmental control chamber (e.g., Lionheart FX)</td><td>Agilent Technologies (or equivalent)</td><td>BioTek Lionheart FX Automated Microscope</td><td>Enables long-term time-lapse imaging under controlled temperature and &lt;sub&gt;2&lt;/sub&gt; conditions</td></tr><tr><td>Imaging software for automated live-cell imaging (e.g., Gen5)</td><td>Agilent Technologies (or equivalent)</td><td>N/A</td><td>Used for acquisition and analysis of time-lapse imaging data</td></tr><tr><td>Centrifuge (swing-bucket rotor)</td><td>Thermo Fisher Scientific (or equivalent)</td><td>Multifuge X4R Pro with TX-1000 rotor (or equivalent)</td><td>Used for PBMC isolation and cell washing steps</td></tr><tr><td>&lt;sub&gt;2&lt;/sub&gt; incubator (37&amp;deg;C, 5% &lt;sub&gt;2&lt;/sub&gt;)</td><td>Thermo Fisher Scientific (or equivalent)</td><td>Forma Direct Heat &lt;sub&gt;2&lt;/sub&gt; Incubator (or equivalent)</td><td>Maintains physiological conditions for cell culture</td></tr><tr><td>&lt;sub&gt;2&lt;/sub&gt; gas supply with regulator</td><td>Agilent Technologies (or equivalent)</td><td>N/A</td><td>Supplies &lt;sub&gt;2&lt;/sub&gt; for incubator and imaging system environmental control</td></tr><tr><td>Conical tubes (15 mL and 50 mL)</td><td>Corning (or equivalent)</td><td>352070 (or equivalent)</td><td>Used for PBMC isolation, washing, and centrifugation steps</td></tr><tr><td>EDTA-coated blood collection tubes (vacutainers)</td><td>BD Biosciences (or equivalent)</td><td>367863 (or equivalent)</td><td>Used for collection of whole blood samples from donors</td></tr><tr><td>Ficoll-Paque PLUS</td><td>Cytiva (or equivalent)</td><td>17144003 (or equivalent)</td><td>Density gradient medium for isolation of PBMCs</td></tr><tr><td>Flow cytometer (e.g., FACSCanto II)</td><td>BD Biosciences (or equivalent)</td><td>BD FACSCanto II&amp;nbsp;</td><td>Used for phenotypic analysis of macrophages</td></tr><tr><td>Compensation beads (flow cytometry)</td><td>Biolegend (or equivalent)</td><td>424602</td><td>Used for single-color compensation controls</td></tr><tr><td>FITC anti-human CD11b antibody (flow cytometry)</td><td>BioLegend (or equivalent)</td><td>101205 (or equivalent)</td><td>Marker used to identify macrophages</td></tr><tr><td>PE anti-human CD80 antibody (flow cytometry)</td><td>BioLegend (or equivalent)</td><td>375410 (or equivalent)</td><td>Marker associated with activated macrophage phenotype</td></tr><tr><td>GFP-expressing breast cancer cell line (MDA-MB-468)</td><td>ATCC (or equivalent)</td><td>HTB-132 (or equivalent)</td><td>Target cells used in phagocytosis assay; GFP enables visualization</td></tr><tr><td>ImmunoCult-SF macrophage medium</td><td>STEMCELL Technologies (or equivalent)</td><td>10961 (or equivalent)</td><td>Serum-free medium used for macrophage differentiation</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher Scientific (or equivalent)</td><td>12100046</td><td>Used for culture and resuspension of cancer cells</td></tr><tr><td>Isotype control antibody (IgG)</td><td>Bio X Cell (or equivalent)</td><td>BE0297 (or equivalent)</td><td>Negative control for antibody-dependent phagocytosis assay</td></tr><tr><td>Lipopolysaccharide (LPS)</td><td>Sigma-Aldrich (or equivalent)</td><td>L2630 (or equivalent)</td><td>Stimulus used to activate macrophages in combination with IFN-&amp;gamma;</td></tr><tr><td>Recombinant human interferon gamma (IFN-&amp;gamma;)</td><td>STEMCELL Technologies (or equivalent)</td><td>78020 (or equivalent)</td><td>Cytokine used for macrophage activation</td></tr><tr><td>Recombinant human macrophage colony-stimulating factor (M-CSF)</td><td>STEMCELL Technologies (or equivalent)</td><td>78057 (or equivalent)</td><td>Growth factor used for macrophage differentiation</td></tr><tr><td>Pasteur pipettes or serological pipettes</td><td>Jetbiofil (or equivalent)</td><td>GSP010010 (or equivalent)</td><td>Used for liquid handling during PBMC isolation and cell culture</td></tr><tr><td>PBS (Ca&lt;sup&gt;2+&lt;/sup&gt;-/Mg&lt;sup&gt;2+&lt;/sup&gt;-free)</td><td>Thermo Fisher Scientific (or equivalent)</td><td>BP399-500 (or equivalent)</td><td>Buffer used for washing cells and dilution steps</td></tr><tr><td>Petri dishes (100 mm, non&amp;ndash;tissue culture-treated)</td><td>Corning (or equivalent)</td><td>430591</td><td>Used for macrophage differentiation via adherence selection</td></tr><tr><td>Red blood cell (RBC) lysis buffer</td><td>BioLegend (or equivalent)</td><td>420301 (or equivalent)</td><td>Used to remove red blood cells during PBMC isolation</td></tr><tr><td>Viobility 405/452 Fixable Dye</td><td>Miltenyi Biotec (or equivalent)</td><td>130-130-404 (or equivalent)</td><td>Used to assess cell viability during flow cytometry analysis</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich (or equivalent)</td><td>D9542 (or equivalent)</td><td>Used for dead cell exclusion in flow cytometry</td></tr></tbody></table>

time-lapse imaging of antibody-driven macrophage phagocytosis of green fluorescence protein&#8211;labeled triple-negative breast cancer cells

Time-Lapse Imaging of Antibody-Driven Macrophage Phagocytosis of Green Fluorescence Protein&#8211;Labeled Triple-Negative Breast Cancer Cells

Antibody-dependent cellular phagocytosis is a key mechanism by which macrophages engulf antibody-opsonized cancer cells. This protocol describes a time-lapse live-cell imaging method to visualize antibody-dependent macrophage phagocytosis of green fluorescent protein&#8211;labeled, CD24-expressing triple-negative breast cancer cells. Macrophages are derived from human peripheral blood mononuclear cells, differentiated with macrophage colony&#8211;stimulating factor, and polarized using interferon gamma and lipopolysaccharide to generate a pro-inflammatory phenotype. Cancer cells are opsonized with an anti-CD24 monoclonal antibody prior to co-culture with macrophages at a defined effector-to-target ratio. Phagocytic events are recorded over a 16 h period using an automated microscope equipped with environmental control to maintain physiological conditions. Imaging is performed using brightfield and fluorescence channels, allowing identification of cancer cells and monitoring of their internalization by macrophages. Representative time-lapse images demonstrate the progressive engulfment of fluorescent cancer cells over time. To enable quantitative assessment, a parallel endpoint assay is performed under identical co-culture conditions, in which non-internalized cancer cells are removed prior to imaging to allow accurate measurement of phagocytic events. This method provides a standardized and reproducible approach for visualizing antibody-driven macrophage phagocytosis in vitro. Importantly, the integration of time-lapse imaging with endpoint-based quantification enables complementary evaluation of the kinetics and magnitude of phagocytosis. This protocol supports the evaluation of therapeutic antibodies and macrophage-directed cancer immunotherapies in a controlled in vitro setting.

Successful generation and polarization of macrophages from PBMCs were confirmed using morphological assessment and flow cytometry analysis (Figure 1). PBMCs isolated by density gradient centrifugation were cultured in M-CSF-supplemented medium, resulting in adherent cells with characteristic macrophage morphology by Day 7. Following stimulation with IFN-&#947; and LPS, cells exhibited a phenotype consistent with activation under the applied conditions. Flow cytometry analysis demonstrated th...

This protocol describes a time-lapse live-cell imaging method to visualize antibody-dependent macrophage phagocytosis of green fluorescent protein&#8211;labeled triple-negative breast cancer cells using peripheral blood mononuclear cell&#8211;derived macrophages and an automated microscope.

Antibody-dependent cellular phagocytosis is a key mechanism by which macrophages engulf antibody-opsonized cancer cells. This protocol describes a ...

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Research

JoVE Journal

Immunology and Infection

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Here, we present a protocol for intravital imaging of an attenuated BSL-2-approved Mycobacterium tuberculosis. strain in the murine lung using a permanent lung window. This approach enables longitudinal, single-cell-resolution analysis of early host-pathogen interactions, including bacillary localization, vascular dynamics, and macrophage uptake within the same lung microenvironment.

Intravital Imaging of Attenuated Auxotrophic Mycobacterium tuberculosis in Murine Lung during Early Intravascular Infection

National Taiwan University Hospital.  This protocol describes a time-lapse live-cell imaging method to visualize antibody-dependent macrophage phagocytosis of green fluorescent protein–labeled triple-negative breast cancer cells using peripheral blood mononuclear cell–derived macrophages and an automated microscope.

Time-Lapse Imaging of Antibody-Driven Macrophage Phagocytosis of Green Fluorescence Protein–Labeled Triple-Negative Breast Cancer Cells

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