Name: in vitro quantitative imaging assay for phagocytosis of dead neuroblastoma cells by ipsc-macrophages
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Description: Watch this Scientific Journal Video about In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages at JoVE.com

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.

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;L, 20-200 &#181;L, 100-1000 &#181;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 &#956;M, by dilution of DMSO stock with Macrophage media.&#160;</td>
		</tr>
		<tr>
			<td>Class 2 laminar air flow safety cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 gas bottle</td>
			<td></td>
			<td></td>
			<td>Accessory for EVOS FL Auto</td>
		</tr>
		<tr>
			<td>CO2 incubator, set to 37&#176;C and 5 % CO2</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&#176;C, avoid further freeze-thaw cycles. Use at final concentration 10 &#181;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&#8217;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 &#956;L (12.5 &#956;g) of pHrodo iFL Red STP Ester stock to pre-warmed cells suspended in Live Cell Imaging Solution.&#160;</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 &#34;Factories&#34;</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&#176;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. ...

Research

JoVE Journal

Neuroscience

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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

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 ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...