analysis of efferocytosis of apoptotic thymocytes by peritoneal macrophages

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

To harvest thymocytes after opening the chest cavity of a na&#239;ve C57/Black-6 mouse, use curved fine-tip forceps to pull out the thymus. Place the organ into a tissue culture dish containing 10 milliliters of RPMI-1640 medium. Grind the whole thymus against the frosted ends of 2 microscope slides, and filter the resulting tissue suspension through a 70-micrometer cell strainer into a 50-milliliter tube. Collect the thymocytes by centrifugation, and resuspend the pellet in 40 milliliters.
After counting, centrifuge the cells again, and resuspend up to 2 x 108 cells in 20 milliliters of fresh PBS per 50-milliliter tube. Label the cells with 5-micromolar CFSE in 20 milliliters of PBS per tube. Invert the tubes two to three times to mix, before incubating the thymocytes for no more than 2 minutes at room temperature, protected from light. At the end of the incubation, stop the reaction with 10 milliliters of heat-inactivated horse serum, and collect the cells by centrifugation.
Resuspend the labeled cells in 40 milliliters of fresh PBS for counting and centrifugation, followed by an additional wash with 40 milliliters of medium. After the medium wash, resuspend the thymocytes at a 7 x 106 cells per milliliter of tissue culture medium concentration, and seed the cells in a 100-millimeter tissue culture dish. Then, add staurosporine to the cell culture at a 1-micromolar final concentration, and place the plate in a tissue culture incubator for 4 hours.
For peritoneal macrophage isolation, inject two C57/Black-6 mice intraperitoneally with 1 milliliter of 3% aged thioglycolate at day 0, before opening the abdominal skin without disturbing the peritoneum on day 5.
To flush the peritoneal cavity, use a 10-milliliter syringe equipped with an 18-gauge needle to quickly push 10 milliliters of wash buffer into the cavity before slowly aspirating the wash buffer for collection into a 50-milliliter tube. Wash the peritoneal gavage two times with PBS. Resuspend the peritoneal macrophages at a 2 times 10 to the sixth cells per milliliter of tissue culture medium concentration.
Next, seed 500 microliters of macrophages into each well of a 24-well plate, and place the plate in a tissue culture incubator for 2 hours. At the end of the incubation, aspirate the supernatant to remove the floating cells, and add 500 microliters of tissue culture medium to each well.
Immediately add 0 to 12 x 106 thymocytes in 500 microliters of medium to each well of the culture, and place the plate in the tissue culture incubator for 4 hours. At the end of the incubation, wash each well two times with fresh PBS to remove any free-floating apoptotic cells, followed by a single wash with staining buffer. Next, label the plate-bound macrophages with 200 microliters of staining buffer supplemented with an appropriate anti-CD11b antibody per well, and place the plate at 4 degrees Celsius for 20 minutes.

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1. Preparation of CFSE-labeled apoptotic thymocytes
<ol>
	<li>Euthanize two na&#239;ve C57/B6 mice by CO2 inhalation for 10 min and dissect to open the chest cavity, remove (pull out) the thymus with curved fine-tip forceps into tissue culture petri dish containing 10 mL of RPMI1640 medium.</li>
	<li>Obtain single-cell suspension by grinding the whole thymus against two frosted ends of the microscope slides and then filter the suspension through a 100 &#956;m cell strainer.</li>
	<li>Collect the 10 mL thymus suspension into a 50 mL tube and centrifuge at 300 x g for 5 min.</li>
	<li>Remove the supernatant, resuspend in 40 mL of 1x PBS, and count cell numbers with a hemocytometer.</li>
	<li>Centrifuge at 300 x g for 5 min and remove the supernatant.</li>
	<li>Resuspend in 20 mL of 1x PBS in a 50 mL tube as a single cell suspension with up to 2 x 108 cells (if more than 2 x 108 cells, resuspend the rest of the cells in another 20 mL of 1 x PBS in a different 50 mL tube).</li>
	<li>Make 5 &#956;M of CFSE in equal volume (20 mL) of 1x PBS in a separate 50 mL tube by pipetting 40 &#956;L of CFSE stock solution (2.5 mM) into 20 mL of 1x PBS and mix well by inverting the tube 2 - 3 times.</li>
	<li>Add the 20 mL of CFSE from step 1.7 into the 20 mL of cell suspension from step 1.6 (the final concentration of CFSE is now 2.5 &#956;M). 
	NOTE: For every 20 mL cell suspension, a 20 mL CFSE tube is needed.</li>
	<li>Invert the cell and CFSE mixture tube 2 - 3 times and incubate the mixture in the dark at room temperature for a maximum of 2 min, then stop the reaction by adding 10 mL of heat-inactivated horse serum.</li>
	<li>Centrifuge the 50 mL tube mixture at 300 x g for 5 min at room temperature. 
	NOTE: If the CFSE labeling is successful, the cell pellet will become light yellow.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 40 mL of 1x PBS and count cell numbers with a hemocytometer.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min and discard the supernatant.</li>
	<li>Wash the cell pellet again with 40 mL of RPMI1640 medium.</li>
	<li>Remove the supernatant and resuspend cells with RPMI1640 tissue culture medium (RPMI1640, 20 mM HEPES, 10% FBS (heat inactivated), 20 mM glutamine, and 1x Pen/Strep) at a concentration of 7 x 106 cells/mL in a 100 mm tissue culture dish. 
	NOTE: If more cells are obtained, a separate 100 mm tissue culture dish will be needed.</li>
	<li>Add staurosporine into the cell suspension culture at a final concentration of 1 &#956;M and culture for 4 h at 37 &#176;C in a tissue culture incubator supplied with 5% CO2.</li>
</ol>
2. Preparation of peritoneal macrophages
<ol>
	<li>Inject two C57B6 mice (or any gene-manipulated mice in the lab) intraperitoneally with 1 mL of 3% aged thioglycollate at day 0.</li>
	<li>Euthanize the mice on day 5 as in step 1.1, cut and peel open the abdominal skin but leave the peritoneum intact. Flush the peritoneal cavity by quickly pushing 10 mL of wash buffer (RPMI1640, 2% FBS, 0.04% EDTA) into the peritoneal cavity using a 10 mL syringe attached to an 18 G needle.</li>
	<li>Retrieve the wash buffer slowly with the same needle/syringe and collect the wash buffer into a 50 mL tube.</li>
	<li>Wash the peritoneal gavage twice with 1x PBS, resuspend the peritoneal macrophages in RPMI1640 tissue culture medium at a density of 2 x 106 cells/mL and aliquot 500 &#956;L into each well of the 24-well plate. Leave the plate in a tissue culture incubator for 2 h.</li>
	<li>Remove the floating cells by aspirating and replacing them with 500 &#956;L of fresh culture medium, twice.</li>
	<li>Optionally, add TAM receptor tyrosine inhibitor, RXDX-106, at concentrations indicated in the figure legends into each well of the macrophage culture and incubate for another 2 h.</li>
</ol>
3. Co-culture of peritoneal macrophages with apoptotic thymocytes
<ol>
	<li>Collect apoptotic thymocytes from step 1 and wash three times with RPMI1640 medium. The efficiency of apoptotic induction can be measured at this stage with the Annexin V/7-AAD kit.</li>
	<li>Distribute 0 - 12 x 106 cells (in 500 &#956;L medium) into each well of the macrophage cultures from protocol #2, according to the experimental arrangement (e.g., see Figure 1). This makes the whole culture volume of 1 mL in each well of the 24-well plate. Add the blocking antibody into the culture immediately before the addition of apoptotic thymocytes.</li>
	<li>Culture the cell mixture at 37 &#176;C for 4 h in a tissue culture incubator supplied with 5% CO2.</li>
	<li>Wash each well of the culture with 1x PBS (containing 500 &#956;M EDTA) twice to remove the free-floating apoptotic cells.</li>
	<li>Stain the plate-bound macrophages with CD11b-PE at this stage.
	<ol>
		<li>Wash the plate-bound macrophages with a staining buffer (1x PBS, 1% BSA) once.</li>
		<li>Add 200 &#956;L of staining buffer containing 2 &#956;L CD11b-PE into each well.</li>
		<li>Incubate the plate at 4 &#176;C for 20 min.</li>
		<li>Wash each well of the plate three times with the staining buffer.</li>
		<li>Add 200 &#956;L of staining buffer and proceed with the plate for image analysis under the fluorescent microscope.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ack lysing buffer</td>
			<td>GIBCO</td>
			<td>A10492</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V/7-AAD</td>
			<td>BD Pharmingen</td>
			<td>559763</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mer antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF591</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-PE (clone M1/70)</td>
			<td>BD Pharmingen</td>
			<td>553311</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Invitrogen</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D-2650</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 mM)</td>
			<td>GIBCO</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352017</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum (Heat-inactivated)</td>
			<td>Invitrogen</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 1x</td>
			<td>Corning</td>
			<td>21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Corning</td>
			<td>10040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>RXDX-106</td>
			<td>Selleck Chemicals</td>
			<td>CEP-40783</td>
			<td></td>
		</tr>
		<tr>
			<td>Staurosprine (100mg)</td>
			<td>Fisher Scientific</td>
			<td>BP2541-100</td>
			<td>Add 214.3 ml of DMSO into 100mg to make 1mM stocking solution</td>
		</tr>
	</tbody>
</table>

Source: Zhen, Y. et al., <a href="https://app.jove.com/v/59731">Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.

<img alt="Figure 1" src="/files/ftp_upload/21786/21786_Figure1.jpg" /> 
Figure 1. Percentage of phagocytosis of apoptotic thymocytes by peritoneal macrophages. Apoptotic thymocytes were induced by incubating with 1 &#956;M of staurosporine for 4 h and added into macrophage culture at a ratio as indicated in the figure panels: (A) 1:0; (B) 1:1; (C) 1:2; (D) 1:4; (E) 1:6; (F) 1:8; (G) 1:10; (H) 1:12. Data were acquired with a flow cytometer. The percen...

Source: Zhen, Y. et al., Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages. J. Vis. Exp. (2019)
This video demonstrates a procedure ...

Efferocytosis is the process by which phagocytic cells clear apoptotic cells.
To analyze efferocytosis, obtain apoptosis-susceptible mouse thymocytes. Incubate with cell-permeable CFSE dye.
Intracellular esterases cleave CFSE to cell-impermeable fluorescent dye, staining thymocytes. Add heat-inactivated serum to prevent excess staining.
Plate the stained thymocytes on a culture plate. Add staurosporine, a protein kinase inhibitor, which disrupts the critical cellular signaling cascade, inducing apoptosis. Further, phosphatidylserine residues translocate to the cell membrane&#39;s outer leaflet, functioning as &#34;eat-me signals.&#34;
Add increasing apoptotic thymocyte concentrations to multi-well plate wells containing activated mouse peritoneal macrophages, which express specific receptor tyrosine kinases.
These kinases bind the exposed phosphatidylserine on apoptotic thymocytes, facilitating their internalization by macrophages.
Post-incubation, remove free-floating apoptotic thymocytes. Add fluorophore-tagged anti-CD11b antibodies, which bind to macrophage CD11b molecules.
Using a fluorescence microscope, observe fluorescent apoptotic thymocytes within differently fluorescent macrophages, indicating efferocytosis.
Wells with higher apoptotic thymocyte concentrations display increased internalized thymocytes.

201 ビュー. 腹膜マクロファージによるアポトーシス性胸腺細胞のエフェロサイトーシスの解析

ビデオ: 腹膜マクロファージによるアポトーシス性胸腺細胞のエフェロサイトーシスの解析 - 実験

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

アポトーシス細胞の食作用アッセイ<em&gt;ショウジョウバエ</em&gt;胚

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

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免疫・感染学

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.

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.

単一細胞の蛍光顕微鏡による Efferocytosis の定量化

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

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

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Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages