This study was funded by Canadian Institutes of Health Research (CIHR) Operating Grant MOP-123419, Natural Sciences and Engineering Research Council of Canada Discovery Grant 418194, and an Ontario Ministry of Research and Innovation Early Research Award to BH. DGW contributed some of the images presented, to the optimization of the protocols and to the writing of the manuscript; he was funded by a pump-priming grant from the university of Liverpool. CY is funded by a Vanier Graduate Scholarship and CIHR MD/PhD Studentship. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Collection of blood from healthy volunteers was approved by the Health Science Research Ethics Board of the University of Western Ontario. Venipuncture was performed in accordance with the guidelines of the Tri-Council Policy Statement on human research.
1. Culture and Preparation of the THP-1 Monocyte Cell Line
<ol>
	<li>Culture THP-1 monocytes as a suspension culture in T25 flasks at 37 &#176;C + 5% CO2. Cells should be grown in 5 mL of Roswell Park Memorial Institute 1640 (RPMI...

The methods outlined in this protocol enable the imaging and quantification of the dynamic efferocytic process, using both fixed-cell and live-cell approaches. These approaches offer several advantages over commonly employed flow cytometry-based methods23,24. The use of inside-out staining with fixed samples provides a more robust and accurate quantification of the rate and extent of efferocytosis &#8212; indeed, many flow cytometry-based methods simply label apo...

Apoptosis, or programmed cell death, is a highly-regulated physiological process that occurs in most multicellular organisms and is crucial for their development and homeostasis1. In addition to being involved in normal cell turnover and embryonic development, apoptosis enables the elimination of infected or damaged cells from tissues and can be triggered in response to infection, inflammation, cancer, and also by medical interventions such as radiotherapy or steroids1. Apoptotic cells expose &#34;eat-me&#34; signals on their cell surface which are recognized by receptors on a range of professional and non-professional phagocytes, collectively referred to as &#34;efferocytes&#34;. Engagement of these receptors induces the uptake and degradation of the apoptotic cell by the efferocyte through a process known as efferocytosis2,3. Phosphatidylserine is the best characterized eat-me signal driving efferocytosis. It is normally confined to the inner leaflet of the plasma membrane, with apoptosis activating a lipid scramblase which disrupts this membrane asymmetry, thus exposing phosphatidylserine on the cell surface4. Phosphatidylserine is found on the extracellular surface of some non-apoptotic cells, such as mature macrophages and activated platelets. However, these cells are not efferocytosed due to the presence of &#34;don&#39;t eat me&#34; signals, such as CD47, on their cell surface5,6,7. Exposed phosphatidylserine is recognized by an array of efferocytic receptors expressed by efferocytes. Binding of these receptors to phosphatidylserine, either directly or through the aid of opsonins, activates signaling pathways that promote the engulfment of the apoptotic cell into a membrane-bound vacuole termed the efferosome8,9,10,11,12. The efferosome fuses sequentially with endosomes and lysosomes, which deliver the molecular machinery necessary to acidify the efferosome and to degrade the apoptotic cell cargo13,14. Once degraded, the apoptotic cell-derived materials are trafficked to the recycling endosome &#8212; a process which limits immune responses to apoptotic cell-derived antigens, and which may allow for recovery of nutrients from the apoptotic cell13,15. A failure in efferocytosis results in impaired clearance of apoptotic cells; these uncleared cells eventually undergo secondary necrosis. Necrotic cells release pro-inflammatory cytosolic contents, pathogens, and autoantigens into the extracellular milieu, thus driving a range of infective, inflammatory and autoimmune diseases16,17. Together, apoptosis and efferocytosis facilitate the removal of dying and dead cells and allow for the maintenance of tissue homeostasis.
Investigating the molecular mechanisms underlying efferocytosis requires methods that provide a clear quantification of apoptotic cell uptake. This quantification is complicated by the fact that unlike other uptake mechanisms such as endocytosis and phagocytosis18,19, efferocytosis may not result in the engulfment of intact target cell, resulting in the piecemeal uptake of the apoptotic cell by the efferocyte20. The protocol described herein describes an in vitro efferocytosis assay that provides accurate delineation of the internalized versus non-internalized portions of individual apoptotic cells and can be combined with a variety of fixed-cell and live-cell microscopy approaches. Traditional phagocytosis assays add antibodies specific to the phagocytic target at the end of the experiment in order to label non-internalized targets, where as our method differs by labelling the apoptotic target with covalently-linked biotin21,22. While apoptotic cell specific antibodies can be used in this assay, the biotinylation approach allows for any protein-bearing target to be labeled and avoids potential issues with secondary antibody cross-reactivity if immunostaining is performed. Specifically, we outline the preparation of apoptotic Jurkat cells that have been dual-stained with both a cell tracking dye and biotin. The cell tracking dye allows for apoptotic cell-derived materials to be tracked during efferocytosis, whereas surface biotinylation allows for the discrimination of internalized from non-internalized portions of efferocytosed apoptotic cells. We also describe the culture and preparation of J774.2 and THP-1 cell lines for use as murine and human efferocytes, monocyte-derived M2 macrophages as an example of primary cell efferocytosis, and Jurkat cells for use as efferocytic targets. These methods can easily be applied to other cell lines or primary cells, to target cells undergoing any form of cell death (e.g. apoptosis, necrosis and necroptosis), and to micron-sized mimics which simulate apoptotic cells through lipid coatings or coating with ligands specific to an efferocytic receptor of interest.
The method outlined in this protocol has several advantages over the flow cytometry based methods commonly used in the field23,24. By directly imaging the phagocyte-apoptotic cell interaction, combined with clear labeling of both total and non-internalized apoptotic cell material, quantitative measures of efferocytosis can be made. Moreover, the use of pH-insensitive fluorophores limits confounding factors such as the suppression of FITC and GFP fluorescence at lysosomal pH that confounds some alternative methods25. Lastly, while not described in detail, these methods can be employed using efferocytes expressing fluorescently-labeled transgenes, or with post-fixation immunostaining, to allow for quantification of signaling molecule activity and monitoring of the cellular processes during efferocytosis.

<table>
	<tbody>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>Wisent</td>
			<td>3500-000-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Wisent</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wisent</td>
			<td>311-010-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm circular glass coverslips #1.5 thickness</td>
			<td>Electron Microscopy Sciences</td>
			<td>72290-08</td>
			<td>Size and shape of coverslip is not critical, but 18 mm fit into the wells of a standard 12-well plate which simplifies cell culture</td>
		</tr>
		<tr>
			<td>Staurosporine</td>
			<td>Cayman Chemical</td>
			<td>81590</td>
			<td>Dissolve in DMSO at 1 mM (1,000x stock solution)</td>
		</tr>
		<tr>
			<td>Annexin V-Alexa 488</td>
			<td>ThermoFisher</td>
			<td>R37174</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Link NHS-Biotin</td>
			<td>ThermoFisher</td>
			<td>20217</td>
			<td>Store in a dessicator. Do not prepare a stock solution.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace FarRed</td>
			<td>ThermoFisher</td>
			<td>C34572</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Orange</td>
			<td>ThermoFisher</td>
			<td>C34851</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Streptavadin</td>
			<td>ThermoFisher</td>
			<td>SA1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte-poly cell sepration medium</td>
			<td>Cedarlane Labs</td>
			<td>CL5071</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>Peprotech</td>
			<td>200-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-4</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>J774.2 Macrophage Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>85011428-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 Human Monocyte Cell Line</td>
			<td>ATCC</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Jurkat T Cell Line</td>
			<td>ATCC</td>
			<td>TIB-152</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of efferocytosis by single-cell fluorescence microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Overnight culture of Jurkat cells with 1 &#181;M staurosporine results in apoptosis of &#62;95% of cells, which can be confirmed with Annexin V staining (Figure 1). Other cell types can be used for these experiments, although the concentration of staurosporine and the duration of staurosporine treatment will need to be optimized for each cell line. For reliable detection and quantification of efferocytosis, &#62;80% of cells should be apoptotic prior to addin...

Watch this Scientific Journal Video about Quantification of Efferocytosis by Single-cell Fluorescence Microscopy at JoVE.com

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

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

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

This method can be used to answer key questions in the efferocytosis field such as the role of signaling molecules in mediating the uptake of apoptotic cells. The main advantage of this technique is that it provides a clear quantification of apoptotic cell uptake including partial or piecemeal apoptotic cell uptake which isn't always apparent with other methods. Though this method can provide insight into efferocytosis by immune cells it can also be applied to other cell types such as epithelial or cancer cells.Generally, individuals new to this method will struggle because of the difficulty in optimizing microscope acquisition settings. After preparing human macrophages and apoptotic Jurkat cells, use a hemocytometer to count the apoptotic cells. Then transfer a sufficient quantity of apoptotic cells to a 1.5 milliliter microcentrifuge tube.Pellet the Jurkat cells via centrifugation at 500 times g for five minutes. Then resuspend the cells in 500 microliters of PBS. While the Jurkat cells are in the centrifuge, aliquot 10 microliters of DMSO into a new microcentrifuge tube.Next, dissolve a minimal amount of NHS-Biotin in the DMSO. After this, transfer 500 microliters of the apoptotic cell, and PBS suspension to the tube containing DMSO and NHS-Biotin. Then, dilute an appropriate cell-tracking dye according to the manufacturer's instructions.Incubate the cell suspension at room temperature for 20 minutes in the dark. Then, add an equal volume of RPMI 1640 with 10%FBS, and incubate the suspension at room temperature in the dark for an additional five minutes. After this, pellet the cells via centrifugation at 500 times g for five minutes.Then discard the supernatant, and resuspend the stained cells in 100 microliters of RPMI 1640 with 10%FBS. Add 100 microliters of stained cell suspension drop wise to each well of macrophages. Then centrifuge the plate at 200 times g for one minute.Incubate the plate at 37 degrees Celsius with 5%CO2 in a tissue culture incubator. After the appropriate incubation period, remove the cells from the incubator. Then wash the cells twice with one milliliter of PBS.After this, add diluted FITC-conjugated streptavidin to each well. And incubate the plate for 20 minutes in the dark. Wash the cells three times with one milliliter of PBS, and gently shake the samples for five minutes after each rinse.Then fix the cells with 4%paraformaldehyde in PBS for 20 minutes at room temperature. Rinse the cells once with PBS to remove any excess PFA. Mount coverslips on an imaging slide, and transfer the slide to a fluorescence microscope.Capture Z stacks of a sufficient number of cells for quantification. It is critical to set the distance between said sections such that all engulfed apoptotic material is detected. The maximum distance between said sections should be no greater than the focal depth of your objective lens, typically 0.5 to one micrometers.Load one of the Z stacks into the software using the Import feature. Then select Integrated density as a measurement option under the Set Measurements panel. Analysis is easiest to perform if both the streptavidin and cell tracking dye channels are displayed as an overlay.Any object with streptavidin staining anywhere along a circumference should be considered streptavidin positive, and therefore, should not be quantified as streptavidin negative. Once the image is loaded, using the streptavidin and the Freehand, or polygon selection tool, circle all of the cell tracking dye positives streptavidin negative material in a single plane of the Z stack. Then, measure the integrated intensity of the cell tracking dye in the region.In the same Z section use the streptavidin staining, and the Freehand or polygon selection tool to circle all of the cell tracking dye positive streptavidin positive material. Finally, measure the integrated intensity of this region. Using this procedure, more than 95%of Jurkat cells treated with Staurosporine went through apoptosis.In most experiments, the fraction of apoptotic cells that are efferocytosed increased in a time-dependent fashion. When optimizing this protocol for different efferocytic cell types, or different apoptotic cell targets, it is important to test the range of apoptotic cell to efferocytic cell ratios. Usually ratios of 10 to one or 30 to one work the best.This procedure can be easily modified to track signaling molecule activation, or intracellular trafficking by expressing fluorescent reporter genes in the efferocytic cells, thus allowing for these processes to be imaged during efferocytosis.

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12.3K vistas.  University of Western Ontario. This method can be used to answer key questions in the efferocytosis field such as the role of signaling molecules in mediating the uptake of apoptotic cells. The main advantage of this technique is that it provides a clear quantification of apoptotic cell uptake including partial or piecemeal apoptotic cell uptake which isn't always apparent with other methods. Though this method can provide insight into efferocytosis by immune cells it can also be applied to other cell types such as epithel...