The authors thank Dr. Val Millar and Dr. Sohaib Nizami for their assistance with high-content microscopy, and Dr. Daniel Ebner for access to the high-content microscopes. Furthermore, the authors thank Dr. Emma Mead for assay development advice, and Mrs. Cathy Browne for iPSC support. This work was supported by the Alzheimer&#39;s Research UK Oxford Drug Discovery Institute (ARUK ODDI, grant reference ARUK-2020DDI-OX), with additional support to the James Martin Stem Cell Facility Oxford (S.A.C.) from the Oxford Martin School LC0910-004; Monument Trust Discovery Award from Parkinson&#39;s UK (J-1403); the MRC Dementias Platform UK Stem Cell Network Capital Equipment MC_EX_MR/N50192X/1, Partnership MR/N013255/1, and Momentum MC_PC_16034 Awards.

<ol>
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The authors have nothing to disclose.

Microglia have important functions that affect the initiation and progression of neurodegenerative diseases, including phagocytosis of apoptotic neurons. Impaired microglial phagocytosis and inappropriate phagocytosis of synapses have both been associated with neurodegenerative diseases, although the underlying mechanisms and causation are not well-understood4,23. This paper outlines a phagocytosis assay to measure phagocytosis of apoptotic cells by iPSC-macrophages, with either a live-cell time-lapse imaging readout or fixed-cell high-content microscopy, or a combination of both on a single assay. This versatility means that the assay can be used to study individual phagocytic events over time in a few wells or used for high-content screening with multiple conditions or treatments. Since the high-content assay is fixed at a single timepoint, multiple assay plates could be prepared simultaneously. The high-content assay has potential utility for characterizing macrophages/microglia with disease-associated genetic variants or screening small molecule inhibitors for changes to phagocytosis. The assay can also be easily adapted to study phagocytosis of other microglia models, or potentially astrocytes. The phagocytosis assay can potentially be multiplexed with live-cell imaging stains, e.g., mitochondria, calcium, or ROS indicators, and post-fixation immunofluorescent staining for proteins-of-interest can be performed. Compared with existing phagocytosis assays that utilize apoptotic neuronal cells, the main advantages that this protocol confers is that preparation of the phagocytic cargo is relatively simple and rapid, and results in a uniform product. Other assays induce apoptosis of neurons or SH-SY5Ys with S-nitroso-L-cysteine for 2 h25, okadaic acid for 3 h22, staurosporine for 4-16 h26,27,28,29 or UV-irradiation for 24 h30, and can result in cells at different stages of apoptosis. Furthermore, the live-cell imaging and high-content imaging readouts have not previously been described, as far as the authors are aware. The main limitation of using paraformaldehyde-fixation to prepare the phagocytic cargo is that it does not fully recapitulate the process of apoptosis, since fixation prevents the cells from splitting into apoptotic bodies, which are likely to be phagocytosed more rapidly due to their smaller size. It is not known what effect fixation has upon the secretion of nucleotide &#34;find me&#34; signals (e.g., ATP, UDP) from the target cell that attract phagocytes. Similar to apoptotic cells, the fixed SH-SY5Ys exhibit some membrane permeability to propidium iodide. Membrane permeability is associated with the release of &#34;find me&#34; signals; however, this has not been studied in the fixed SH-SY5Ys, and if the nucleotide are released too quickly, they would be washed away before SH-SY5Ys are added to the iPSC-macrophages.
The first critical step in the protocol is staining of dead SH-SY5Ys with an STP ester of a pH-sensitive red fluorescent dye. This dye rapidly and covalently reacts with free primary amines on the surface of the dead SH-SY5Ys. The duration of staining does not need to be optimized; however, care must be taken with handling the dye before labeling. The labeling reaction must not be performed in buffers containing free amines. Furthermore, there is a risk of precipitation if the DMSO stock is diluted in cold aqueous buffer or at high final concentration. Precipitates will appear as dense dark objects under the microscope. Additionally, the pH-sensitive dye solution sticks to regular plastic centrifuge tubes and washes off slowly; therefore, low-binding tubes are recommended for the labeling step. Use of a pH-sensitive dye, instead of a permanently fluorescent dye, aids the identification of engulfed particles, versus particles that neighbor the plasma membrane. Since there is some fluorescence at neutral pH, the density of phagocytic cargo and iPSC-macrophages need to be kept low enough for accurate segmentation, although high enough that numerous phagocytic events are captured. High-content microscopy was capable of accurately identifying phagocytosis with a medium density of cargo in the well (more than 2 SH-SY5Ys per iPSC-macrophage). Conversely, due to weaker sensitivity of the microscope in the deep red spectrum, segmentation of iPSC-macrophages in the live-cell time-lapse imaging data was less confident and it was necessary to use a very low density of cargo to reduce the likelihood of false positives (1 SH-SY5Y for every two iPSC-macrophages). Validation of proper segmentation and cargo density should be performed with comparisons between untreated and cytochalasin D-treated wells. In a well-optimized assay, cytochalasin D should reduce the average number of spots per cell by 90% relative to untreated samples.
Another critical step in the protocol is the iPSC-macrophage staining, which allows the cell to be identified and segmented in image analysis so that any external SH-SY5Ys are excluded from the count. The recommended dye is cell-permeant, converted to an insoluble fluorescent product within the cytoplasm, fixable, and non-toxic (see Table of Materials). The staining step was optimized for use of iPSC-macrophages with the high-content imaging phagocytosis assay, and we suggest that it should be re-optimized if other cell types are used. The duration of cell staining can be increased to improve deposition of the insoluble fluorescent product within cells. If dye concentration is optimized, care should be taken to avoid toxic levels of the organic solvent vehicle.
The third critical factor to the success of the assay is the data analysis. The analysis pipelines provided are intended to be of guidance rather than prescriptive, as differences to staining intensity or cell morphology can reduce the effectiveness of segmentation of the pipelines as written. Some optimizations will therefore be required, with testing of the pipeline on appropriate positive and negative controls, and the parameters that should be optimized are indicated in the protocol text. Negative controls should include a condition where iPSC-macrophages are pre-treated with a potent phagocytosis inhibitor such as cytochalasin D before addition of SH-SY5Ys. Another possible negative control is addition of the SH-SY5Ys to previously untreated wells of iPSC-macrophages at the end of the assay, 10 min before fixation, which allows some settling of the cargo but is too short for an appreciable quantity of phagocytosis to occur. A phagocytosis event is defined as a red-fluorescent object within the borders of an iPSC-macrophage, defined by the software algorithm using the deep red fluorescence channel. If segmentation of the cells is poor (Figure 2), many non-phagocytosed SH-SY5Ys in close proximity to iPSC-macrophages may be erroneously included in the analysis, i.e., false positives. The most important factor in achieving good segmentation is stringent delineation of the iPSC-macrophages. Segmentation for both analyses is automated, so it is not possible to obtain perfect segmentation for every cell; however, a few parameters can be adjusted to make the segmentation more optimal, using a few test images as reference. The cytochalasin D control is important for assessing optimal segmentation because a high number of phagocytic events detected in this condition indicates that the segmentation is sub-optimal. Optimization of the data analysis pipeline should ideally be repeated until the number of phagocytic events per cell is 80%-90% lower in the cytochalasin D condition versus no inhibitor.
The problems with the phagocytosis assay that are most likely to occur are: (1) weak pH-sensitive fluorescence in positive controls, (2) sparse or uneven distribution of macrophages at the end of the assay, or (3) high numbers of false-positives in the analysis from non-phagocytosed SH-SY5Ys. Troubleshooting of weak pH-sensitive fluorescence should firstly check that staining of the SH-SY5Ys resulted in a cell pellet with a strong magenta color. If the color is weak, ensure that a fresh dye stock is used, ensure that the labeling buffer is amine-free, add an extra wash to the SH-SY5Ys before staining, check whether the correct number of SH-SY5Ys were stained, ensure that no dye precipitates are in evidence, and optimize the labeling concentration of the dye. If the SH-SY5Ys are strongly stained, check whether the concentration added to the assay plate is correct, and ensure that the iPSC-macrophages are healthy and not too old. The second type of issue, uneven macrophage distribution, can result from loss of cells during pipetting and steps should be taken to reduce the pipetting forces experienced by the cells, avoiding narrow-bore tips. If the problem remains, reduce the incubation time of loading the iPSC-macrophages with cell-permeant dye. The third problem, regarding erroneous inclusion of non-phagocytosed particles in the analysis, indicates that more optimization of the analysis pipeline is required. Troubleshooting should focus firstly on cell segmentation and whether the software is including adjacent objects. Specific parameters that can be adjusted are suggested in the notes below the relevant steps (steps 6.1.11-6.1.15 for the live-cell time-lapse analysis and steps 6.2.4-6.2.8 for the high-content analysis). If cell segmentation cannot be further improved, the high-content analysis has an extra step (step 6.2.8) that excludes improperly segmented iPSC-macrophages. Furthermore, the module that filters accepted spots of pH-sensitive fluorescence within iPSC-macrophages can be optimized, increasing the threshold intensity of accepted objects, which should help to exclude non-phagocytosed SH-SY5Ys (step 6.1.17 for the live-cell time-lapse analysis, and step 6.2.11 for the high-content analysis).
We developed two types of microscopy readout for the phagocytosis assay that each have advantages and limitations. Live-cell time-lapse imaging has the merits of providing extra information about phagocytosis kinetics and is more widely available than high-content imaging platforms. The recommended open-source software is agnostic to the microscope source and could be used with any good-quality fluorescent microscope, with or without live-cell time-lapse capability. The main limitation of the live-cell imaging is limited sensitivity and optics, which make it more challenging to detect and perform good segmentation of iPSC-macrophages. This limitation could be mitigated either by increasing the duration of iPSC-macrophage staining, or by switching to a more sensitive microscope, if available. The high-content imaging phagocytosis assay is the recommended readout if a high-content imaging system is available. High-content imaging systems enable higher throughput and more reliable data, enabling this assay to be used for screening, in which a robust Z&#39; of &#8805;0.7 would be expected for the &#34;number of spots per cell&#34; output20. Compared with the live-cell time-lapse method, the high-content microscopy readout has higher sensitivity, a higher degree of automation and speed, more wells and imaging fields can be processed, and high-resolution confocal images are produced. Cell segmentation is more effective with good images, and segmentation is additionally aided by the high-content imaging analysis software providing more cell segmentation methods suitable for highly irregularly shaped cells. The high-content imaging analysis software also calculated more parameters of phagocytosis, compared with the open-source software, such as the percentage of phagocytic cells. The main limitation of the high-content phagocytosis assay is one of cost and accessibility of the imaging system and analysis software.
In conclusion, the quantitative phagocytosis assay presented in this paper is a useful tool for modeling microglia phagocytosis of dead neurons in vitro. The microglia are modeled by iPSC-macrophages and the dead neurons are modeled by paraformaldehyde-fixed SH-SY5Ys. Although not the most authentic microglia and dead/apoptotic neuron models published, these are easy to prepare and scalable. The assay itself is highly versatile, with two types of imaging readout detailed, and it has potential to be adapted for use with different microglia/macrophage monoculture models, or a different cell type to act as the phagocytic cargo. The high-content imaging readout is advantageous for obtaining quantitative data and can be scaled up to assay small molecule modulators of phagocytosis, or screen genetic variants in the iPSC-macrophages. However, since high-content imaging systems are expensive and data-heavy, an alternative imaging readout has been included in the protocol using a live-cell time-lapse microscope, which could be substituted for any good-quality conventional fluorescence microscope, if needed.

Microglia are brain tissue-resident macrophages, and their functions include immune surveillance, coordinating inflammatory responses to injury/infection, synaptic remodeling, and phagocytosis of dead cells, myelin, protein aggregates, and pathogens. Phagocytosis is the process by which microglia recognize cargo with surface receptors and reorganize their cytoskeleton to engulf the object into a phagosome, which then fuses with lysosomes for degradation of the cargo. Healthy microglia phagocytose apoptotic brain cells to remove them before they become necrotic1. The phagocytosis of apoptotic cells is also known as efferocytosis, and requires the display of a phosphatidylserine &#34;eat-me&#34; signal by the dying cell2. Numerous microglia receptors directly bind to phosphatidylserine, including TIM-4, BAI1, Stabilin-2, and TREM2. Microglial TAM receptors (e.g., MERTK) and integrins indirectly bind to phosphatidylserine, using accessory proteins GAS6 or MFG-E8, respectively. Other &#34;eat-me&#34; signals may be necessary for recognition of dying cells, these include changes to glycosylation or charge of surface proteins; expression of intracellular proteins ICAM3, calreticulin, annexin-I at the cell surface; oxidized LDL; or coating of the apoptotic cell by microglia-produced complement C1q1,2.
Neurodegenerative diseases, including Parkinson&#39;s disease, Alzheimer&#39;s disease, frontotemporal dementia, and amyotrophic lateral sclerosis have been associated with impairment to microglia function, including an accumulation of brain waste products such as dead cells, myelin fragments, and protein aggregates, and exaggerated inflammatory responses to these stimuli3. Phagocytosis may be impaired in neurodegenerative diseases and contribute to the pathology, due to a combination of aging, inflammation, or specific genetic risk variants4,5. On the other hand, there is also evidence from animal models of neurodegenerative diseases that microglia may inappropriately phagocytose viable neurons or synapses6,7,8. The mechanism is likely to be instigated by phosphatidylserine display of damaged neurites, which is directly sensed by microglial phagocytosis receptors TREM2 or GPR56, or indirectly sensed by soluble complement C1q coating the phosphatidylserine-enriched membrane, leading to CR3-mediated phagocytosis9,10,11.
In vitro assays of phagocytosis function, e.g., to assess the phenotypic impact of a genetic risk variant in microglia, are frequently performed using non-physiological cargoes such as latex beads4. Fluorescently labeled bacteria and zymosan are also used, which are physiological but not relevant to neurodegenerative diseases. Non-physiological phagocytic cargoes can be used to detect defects in the basic machinery of phagocytic engulfment but fail to accurately model the first &#34;recognition&#34; step in phagocytosis of apoptotic neurons. The size, shape, stiffness, and type of cargo also dictate the intracellular signaling pathways that are activated, leading to different outcomes of microglia activation state. For example, E.coli bacteria are small and stiff, unlike human cells, and the lipopolysaccharides on their surface are recognized by Toll-like receptor 4 (TLR4) that activates phagocytosis and pro-inflammatory signaling pathways2,12.
In the context of neurodegenerative disease studies, a more relevant phagocytic cargo would have phosphatidylserine display on mammalian plasma membranes, and would ideally be human and neuronal, to include signals that microglia are likely to encounter. For this phagocytosis protocol, the human neuroblastoma cell line SH-SY5Y was chosen as a neuron model that is easy to culture. Permanent surface phosphatidylserine display was artificially induced by paraformaldehyde, which has previously been shown to cause phosphatidylserine display of platelets13. For the microglia cell model human iPSC-macrophages were used, which mimic the ontogeny and transcriptional profile of human microglia, and are phagocytically competent14,15,16,17. iPSC-macrophages are not the most authentic microglia model available, e.g., they do not mimic microglia morphology; however, one can substitute it for a more authentic monoculture iPSC model of microglia if desired, such as Haenseler et al.15. Human iPSC models are preferable to primary rodent microglia for studying neurodegeneration, due to concerns about the limited overlap of the microglia transcriptional modules observed in human versus mouse neurodegenerative disease tissues18. The dead SH-SY5Ys are stained with an acid-sensitive dye that fluoresces weakly at neutral pH and more strongly inside the phagolysosomes of iPSC-macrophages after phagocytosis. Using an acid-sensitive dye improves the accuracy of detecting phagocytic events, with versatility for different readouts of live and fixed macrophages19. This protocol outlines both live-cell time-lapse imaging of phagocytosis, and a fixed high-content imaging assay for phagocytosis, with the same cell preparation steps prior to readout (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62217/62217fig1v2.jpg" /> 
Figure 1: Schematic diagram of methodology. Outline of the phagocytosis assay, where preparation of the SH-SY5Ys and staining of the iPSC-macrophages is performed in parallel, and then the SH-SY5Ys are pipetted onto the iPSC-macrophages. Either live-cell time-lapse imaging is performed immediately, or the cells are incubated at 37 &#176;C/5% CO2 for the required duration and fixed before performing high-content microscopy. PFA: paraformaldehyde, HBM: phenol red-free HEPES-buffered media, pHr: pH-sensitive red fluorescent dye STP Ester solution, PRFMM: phenol red-free macrophage media. <a href="https://www.jove.com/files/ftp_upload/62217/62217fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>15 mL conical centrifuge tube</td><td>Falcon</td><td>352096</td><td>For centrifugation of cells</td></tr><tr><td>2-20 &amp;micro;L, 20-200 &amp;micro;L, 100-1000 &amp;micro;L single-channel micropipettes</td><td></td><td></td><td></td></tr><tr><td>2-mercaptoethanol 50 mM</td><td>Gibco</td><td>31350010</td><td>Component of Factory media</td></tr><tr><td>4% paraformaldehyde in PBS</td><td>Alfa Aesar</td><td>J61899</td><td>For fixation of cells</td></tr><tr><td>6-well plate, tissue culture treated</td><td></td><td></td><td></td></tr><tr><td>AggreWell-800 24-well plate</td><td>STEMCELL Technologies</td><td>34815</td><td>Microwell low-adherence 24-well plate for formation of embryoid bodies</td></tr><tr><td>Annexin V-FITC Apoptosis Staining / Detection Kit</td><td>Abcam</td><td>ab14085</td><td>Kit for annexin V-FITC staining , as an assay for quality control of fixed SH-SY5Ys. Kit contains annexin binding buffer, annexin V-FITC, and propidium iodide.</td></tr><tr><td>Automated cell counter</td><td></td><td></td><td></td></tr><tr><td>Benchtop centrifuge</td><td></td><td></td><td></td></tr><tr><td>Benchtop microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>CellCarrier-96 Ultra Microplates, tissue culture treated, black, 96-well with lid</td><td>Perkin Elmer</td><td>6055302</td><td>96-well tissue culture (TC)-treated microplate with black well walls and an optically-clear bottom, for phagocytosis assay</td></tr><tr><td>CellProfiler software</td><td></td><td></td><td>Open-source software for analysis of phagocytosis images obtained by live-cell time-lapse microscope. Download for free from website (http://cellprofiler.org/), this protocol used version 2.2.0.</td></tr><tr><td>CellTracker Deep Red dye</td><td>Thermo Fisher</td><td>C34565</td><td>Deep red-fluorescent, cell-permeant, succinimidyl ester-reactive dye for staining cytoplasm of iPS-macrophages. Dissolve CellTracker Deep Red dye in DMSO to 2 mM (1.4 mg/mL). Use at 1 &amp;mu;M, by dilution of DMSO stock with Macrophage media.&amp;nbsp;</td></tr><tr><td>Class 2 laminar air flow safety cabinet</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; gas bottle</td><td></td><td></td><td>Accessory for EVOS FL Auto</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator, set to 37&amp;deg;C and 5 % CO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Columbus Image Data Storage and Analysis System</td><td>Perkin Elmer</td><td>Columbus</td><td>Data storage and analysis platform for Opera Phenix. Supports all major high content screening instruments.</td></tr><tr><td>Cytochalasin D</td><td>Cayman</td><td>11330</td><td>Negative control treatment for phagocytosis assay. Reconstitute in DMSO to 10 mM and store aliquots at -20&amp;deg;C, avoid further freeze-thaw cycles. Use at final concentration 10 &amp;micro;M.</td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11320074</td><td>Component of SH-SY5Y media</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td>Solvent for CellTracker and pHrodo dyes</td></tr><tr><td>EVOS FL Auto Imaging System</td><td>Thermo Fisher</td><td>AMF4300</td><td>Live-cell time-lapse imaging microscope</td></tr><tr><td>EVOS Light Cube CY5</td><td>Thermo Fisher</td><td>AMEP4656</td><td>Accessory for EVOS FL Auto</td></tr><tr><td>EVOS Light Cube DAPI</td><td>Thermo Fisher</td><td>AMEP4650</td><td>Accessory for EVOS FL Auto</td></tr><tr><td>EVOS Light Cube RFP</td><td>Thermo Fisher</td><td>AMEP4652</td><td>Accessory for EVOS FL Auto</td></tr><tr><td>EVOS Onstage Incubator</td><td>Thermo Fisher</td><td>AMC1000</td><td>Accessory for EVOS FL Auto</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F4135</td><td>Component of SH-SY5Y media</td></tr><tr><td>Flow cytometer</td><td></td><td></td><td></td></tr><tr><td>Flow cytometry analysis software</td><td></td><td></td><td></td></tr><tr><td>Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix</td><td>Invitrogen</td><td>A1413302</td><td>hESC-qualified basement membrane matrix for iPSC culture</td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050-038</td><td>Component of both Factory and Macrophage media</td></tr><tr><td>HBSS</td><td>Lonza</td><td>BE 10-547F</td><td>Hank&amp;rsquo;s balanced salt solution for washing steps</td></tr><tr><td>Human recombinant BMP4</td><td>Gibco</td><td>PHC9534</td><td>Component of Embryoid Body media</td></tr><tr><td>Human recombinant IL-3</td><td>Gibco</td><td>PHC0033</td><td>Component of both Factory and Macrophage media</td></tr><tr><td>Human recombinant SCF</td><td>Miltenyi Biotech</td><td>130-096-695</td><td>Component of Embryoid Body media</td></tr><tr><td>Human recombinant VEGF</td><td>Gibco</td><td>PHC9394</td><td>Component of Embryoid Body media</td></tr><tr><td>Live Cell Imaging Solution</td><td>Thermo Fisher</td><td>A14291DJ</td><td>Phenol red-free HEPES-buffered media for labelling dead SH-SY5Ys</td></tr><tr><td>Low protein binding 2 mL tubes</td><td>Eppendorf</td><td>30108.132</td><td>For staining SH-SY5Ys</td></tr><tr><td>M-CSF</td><td>Thermo Fisher</td><td>PHC9501</td><td>Component of both Factory and Macrophage media</td></tr><tr><td>mTeSR1 Medium</td><td>STEMCELL Technologies</td><td>85850</td><td>iPSC media</td></tr><tr><td>Multichannel 20-200 uL pipette</td><td></td><td></td><td>For liquid handling of 96-well plate</td></tr><tr><td>NucBlue Live ReadyProbes Reagent</td><td>Thermo Fisher</td><td>R37605</td><td>Hoechst 33342 formulation in a dropper bottle for staining nuclei of iPS-macrophages, use 0.5 drops/mL in Macrophage media.</td></tr><tr><td>Opera Phenix High-Content Screening System</td><td>Perkin Elmer</td><td>HH14000000</td><td>High-content imaging microscope, used with Harmony software version 4.9.</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Component of Factory, Macrophage, and SH-SY5Y media</td></tr><tr><td>pHrodo iFL Red STP-Ester</td><td>Thermo Fisher</td><td>P36011</td><td>pH-sensitive red fluorescent dye for labelling dead SH-SY5Ys. Reconstitute pHrodo iFL Red STP Ester powder in DMSO to a 5 mg/mL concentration. For each 1 million SH-SY5Ys, add 2.5 &amp;mu;L (12.5 &amp;mu;g) of pHrodo iFL Red STP Ester stock to pre-warmed cells suspended in Live Cell Imaging Solution.&amp;nbsp;</td></tr><tr><td>Serological pipette filler</td><td></td><td></td><td></td></tr><tr><td>T175 flask, tissue culture treated</td><td></td><td></td><td>Vessel for differentiations of iPSC-macrophage precursors, known as &quot;Factories&quot;</td></tr><tr><td>T75 flask</td><td></td><td></td><td>Vessel for SH-SY5Y culture</td></tr><tr><td>Transparent plate sealers</td><td>Greiner Bio-One</td><td>676001</td><td>For assay plate storage and transportation</td></tr><tr><td>TrypLE Express (1X), no phenol red</td><td>Gibco</td><td>12604013</td><td>Cell dissociation buffer containing recombinant trypsin-like enzymes and 1.1 mM EDTA, use neat.</td></tr><tr><td>Water bath, set to 37&amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>X-VIVO 15 Medium with L-glutamine, gentamicin, and phenol red</td><td>Lonza</td><td>BE04-418F</td><td>Component of Factory and Macrophage media</td></tr><tr><td>X-VIVO 15 Medium with L-glutamine; without gentamicin or phenol red</td><td>Lonza</td><td>04-744Q</td><td>Phenol red-free macrophage media, for use in phagocytosis without additives or growth factors</td></tr></tbody></table>

in vitro quantitative imaging assay for phagocytosis of dead neuroblastoma cells by ipsc-macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Live-cell time-lapse imaging was performed using the previously outlined protocol, with wild-type iPSC-macrophages seeded at 20,000 cells per well. Different amounts of SH-SY5Ys were applied (10,000-30,000 per well as estimated from the cell count in step 3.1), and the phagocytosis inhibitor cytochalasin D was pre-incubated (1 h) with some wells, acting as a control to inhibit phagocytosis for each amount of SH-SY5Ys. Imaging commenced 40 min after the addition of SH-SY5Ys and images were captured at 5 min intervals for the next 3 h (data includes the initial 40 min delay). A representative time-lapse video is included in the Supplementary Data, and analyzed quantitative data shown in Figure 3. With the amount of 10,000 SH-SY5Ys per well, the number of phagocytosed particles (spots) per cell increased linearly with time, and was inhibited by approximately 50% by cytochalasin D. The inhibition by cytochalasin D was weaker than anticipated, most likely caused by insufficient technical or biological replicates, as only one well per condition was imaged with three image fields. With higher amounts of SH-SY5Ys per well (20,000 and 30,000), phagocytosis exhibited poor linearity, likely due to poor segmentation of iPSC-macrophages and SH-SY5Ys in a more crowded field of view.
Fixed-cell high-content imaging was performed using the previously-outlined protocol, with wild-type iPSC-macrophages at 20,000 cells per well, several different amounts of SH-SY5Ys (10,000-80,000 per well), and the assay plate was fixed and imaged after 5 h. A representative image of phagocytosis is presented in Figure 4A, and the analyzed data shown in Figure 4B17. Increasing the amount of SH-SY5Ys resulted in a higher number of phagocytosed particles (spots) per cell; however, a doubling of SH-SY5Y quantity only leads to a 1.5x increase in number of spots per cell. This indicates that the amounts tested are not rate-limiting to phagocytosis. Subsequently the high-content imaging phagocytosis assay was validated using several inhibitors of phagocytosis (Figure 4C)17. The actin polymerization inhibitors cytochalasin D and jasplakinolide significantly inhibited phagocytosis by 91% and 90%, respectively, when pre-incubated for 1 h prior to phagocytosis. The robust Z&#39; of the assay when cytochalasin D or jasplakinolide are used as negative controls is calculated as 0.7 and 0.8, respectively20. The lysosome acidification inhibitor bafilomycin A1 significantly reduced phagocytosis by 31%, when incubated 1 h prior to phagocytosis. The weaker effect of the lysosome acidification inhibitor versus actin inhibitors suggest that detection of the internalized cargo may not require full acidification of the phagosome. Recombinant annexin V was used as a control to specifically block phosphatidylserine exposed on the surface of SH-SY5Ys, preventing phagocytic receptors from accessing the ligand, an important &#34;eat-me&#34; signal. Addition of recombinant annexin V significantly reduced phagocytosis by 30%, when added to wells immediately before SH-SY5Y addition. Fixed SH-SY5Ys were confirmed to expose phosphatidylserine, using a fluorescent annexin V probe, whereas live SH-SY5Ys were negative for annexin V staining (Figure 4D).
The microglial phagocytosis receptor TREM2 has previously been shown to be important for phagocytosis of apoptotic neurons21. The R47H mutation of TREM2 is a risk gene for late-onset of Alzheimer&#39;s disease, and is hypothesized to reduce ligand binding of TREM223. With the aim of assessing phagocytic function of R47H TREM2 and TREM2 KO, the fixed-cell high-content phagocytosis assay was performed using isogenic iPSC-macrophage lines with WT/R47H/KO TREM217. Several lengths of phagocytosis duration from 1 to 5 h were tested, using a staggered addition of phagocytic cargo (40,000 SH-SY5Ys). The resulting signal increases linearly to 4 h, leveling off slightly at 5 h (Figure 5)17. Reduced phagocytosis rate and capacity (% spot positive cells) was evident in the TREM2 KO compared to WT, whereas the R47H TREM2 mutant did not show altered phagocytosis. The phagocytic defect in TREM2 KO cells is not phenocopied by the R47H TREM2 mutation, seemingly because the TREM2 function is sufficient to support normal phagocytosis.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62217/62217fig3v2.jpg" /> 
Figure 3: Example data for live-cell time-lapse phagocytosis assay. Uptake of dead SH-SY5Ys by wild-type iPSC-macrophages BIONi010-C (ECACC ID: 66540023) imaged at 5 min intervals for 3 h. Times displayed on the graph are from initiation of phagocytosis, including the first 40 min without measurement. The average number of spots per cell from three replicate wells are plotted. Phagocytosis of 10,000 SH-SY5Ys is inhibited with 10 &#181;M cytochalasin D with 1 h pre-treatment, whereas higher amounts of SH-SY5Ys (20,000 and 30,000) have suboptimal quantification of phagocytosis. Mean &#177; standard deviation (SD), N = 1 experiment. <a href="https://www.jove.com/files/ftp_upload/62217/62217fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62217/62217fig4v2.jpg" /> 
Figure 4: Optimization and validation of fixed-cell high-content phagocytosis assay. (A) Representative high-content microscopy image of SH-SY5Ys phagocytosed by wild-type iPSC-macrophages BIONi010-C (ECACC ID: 66540023). A 3 h time-point with 40,000 SH-SY5Ys is shown. Fluorescence channels are merged, with iPSC-macrophage stain shown as red, nuclei as blue, and SH-SY5Ys as yellow. The inset panel is a section of the image magnified 3x. (B) The number of spots per cell of phagocytosed dead SH-SY5Ys after 5 h, using different amounts of cargo addition to wild-type iPSC-macrophages. Mean &#177; standard error of the mean (SEM), for N = 3 harvests. (C) Phagocytosis (3 h) is inhibited with 10 &#181;M cytochalasin D (Cyt), 1 &#181;M bafilomycin A1 (Baf), 1 &#181;M jasplakinolide (with 1 h pre-treatment; Jas), and 13 &#181;g/mL recombinant annexin V (added simultaneously to the dead SH-SY5Ys; Ann). iPSC-macrophages with no SH-SY5Ys added were used as a negative control (-ve), and the positive (+ve) control is untreated iPSC-macrophages with SH-SY5Ys added. Data was normalized to the mean for the experiment repeat. Means &#177; SEM, for N = 3-6 harvests and with two wild-type cell lines (SFC840-03-03, the characterization of this line is described in (Fernandes et al.21 and BIONi010-C). 1-way ANOVA with Dunnett&#39;s post-hoc test, comparisons to untreated cells. *p &#60; 0.05, ***p &#60; 0.001. (D) Freshly fixed SH-SY5Ys stain uniformly for phosphatidylserine display (annexin V-FITC) and have limited cell permeability (propidium iodide). Live SH-SY5Ys do not stain for annexin V-FITC or propidium iodide, except for focal staining present on the few dead cells in culture. Figures are reproduced with permission from Alzheimer&#39;s Research &#38; Therapy17. <a href="https://www.jove.com/files/ftp_upload/62217/62217fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62217/62217fig5v2.jpg" /> 
Figure 5: Phagocytosis is reduced in TREM2 KO but not in R47H TREM2 iPSC-macrophages. High-content phagocytosis assay performed with 40,000 SH-SY5Ys per well with staggered additions. Means were quantified for the parameters: number of spots per cell, sum of spot areas (&#181;m2) per cell, and percentage of cells containing phagocytosed particles per field. Data was normalized to mean for each genotype per experiment. Mean &#177; SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, Dunnett&#39;s post-hoc test, pairwise comparisons to the WT for each time: *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001. Figures are reproduced with permission from Alzheimer&#39;s Research &#38; Therapy17. <a href="https://www.jove.com/files/ftp_upload/62217/62217fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1: Example QC for SH-SY5Ys preparation. Dissociated SH-SY5Ys were fixed for 10 min with 0% (live cells), 1%, and 2% of paraformaldehyde (PFA), then washed. Cells were stained with annexin V-FITC and propidium iodide (PI) and immediately measured by flow cytometry. Color density dot plots were created in flow cytometry analysis software, using the single-stained and unstained controls to place a quadrant gate. Quadrants are annotated with the percentage of events within that quadrant. Live cells are mainly in Q4, and fixed cells are mainly in Q2. Q1 = annexin V-/PI-, Q2 = annexin V+/PI+, Q3 = annexin V+/PI-, Q4 = annexin V-/PI- (live cells). <a href="https://www.jove.com/files/ftp_upload/62217/Supplementary Figure S1.pdf" target="_blank">Please click here to download&#160;this File.</a>
Supplementary Video: Live-cell time-lapse phagocytosis. Representative time-lapse video of SH-SY5Ys phagocytosed by wild-type iPSC-macrophages BIONi010-C (ECACC ID: 66540023). Images were taken every 5 min for 3 h. Video is cropped and runs at 3 frames per second, showing the last 1.5 h of the assay. The acid-sensitive dye-stained SH-SY5Ys are shown in red, signal intensity increasing with phagosome acidification. Cell nuclei stained with Hoechst 33342 are shown in blue. <a href="https://www.jove.com/files/ftp_upload/62217/Supplementary Video.avi" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages at JoVE.com

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

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Neuroscience

5.4K Views.  University of Oxford. Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Video: In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Cancer Research

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Immunology and Infection

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

Bioengineering

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

Biology

Microglial Phagocytosis Assay: An In Vitro Assay to Establish Microglial Phagocytosis Model for Neuroblastoma Cells Using iPSC Macrophages

Source: Hall-Roberts, H. et al. <a href="https://www.jove.com/v/62217/in-vitro-quantitative-imaging-assay-for-phagocytosis-dead">In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages.</a> J. Vis. Exp. (2021)
In this video, we perform the microglial phagocytosis assay for neuroblastoma using induced pluripotent stem cells. The assay helps assess the impairment of microglial function in neurodegenerative diseases.

Source: Hall-Roberts, H. et al. In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages. J. Vis. Exp. (2021)
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JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vitro studies

Live-cell Phagocytosis Assay of Microglia-Like Cells Using Human Synaptosomes

<a href='https://app.jove.com/v/64323/human-microglia-like-cells-differentiation-from-induced-pluripotent'>Source: Funes, S., et. al.,&#160; Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes. J. Vis. Exp. (2022)</a>This video demonstrates an assay to study the phagocytic capacity of microglia-like cells using pH-sensitive fluorescent dye-labeled synaptosomes.

Source: Funes, S., et. al.,&#160; Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Summary

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Chapters in this video

In Vitro Assay to Study Tumor-macrophage Interaction

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Microglial Phagocytosis Assay: An In Vitro Assay to Establish Microglial Phagocytosis Model for Neuroblastoma Cells Using iPSC Macrophages

Live-cell Phagocytosis Assay of Microglia-Like Cells Using Human Synaptosomes