The authors would like to thank Prof. Naiming Zhou from Zhejiang University for his technical advice and for making the equipment of his laboratory available for use. This work was financially supported by the National Natural Science Foundation of China (grant numbers 41876154, 41606150, and 41406137) and the Fundamental Research Funds for Zhejiang Provincial Universities and Research Institutes [grant number 2019JZ00007].

1. Cell Culture Medium Preparation
<ol>
	<li>Coelomic fluid preparation
	<ol>
		<li>Coelomic fluid collection: Under sterile conditions, dissect a healthy sea cucumber (wet weight of 85-105 g), collect coelomic fluid, and store it in a sterile glass flask.</li>
		<li>Coelomic cell removal: Centrifuge the coelomic fluid in 50 mL centrifuge tubes at 1,700 x g for 5 min and transfer the supernatant into a new sterile glass flask; next, collect the cell-free coelomic fluid of the sea cucumber.</li>
		<li>Complement components inactivation: Incubate the sterile glass flask, containing the sea cucumber coelomic fluid, in a 40&#8211;50 &#176;C water bath for 20&#8211;40 min to obtain complement components-inactivated coelomic fluid.</li>
		<li>Microbe removal: Remove bacteria and chlamydia by filtration through 0.22 &#956;m membrane filters. Next, remove mycoplasma and other fine particles by filtration using 0.1 &#956;m membrane filters to obtain coelomic fluid pretreatment solution.</li>
		<li>Salinity adjustment: Adjust the salinity of the coelomic fluid pretreatment solution to 30&#8240; (measured by salinometer) by adding 20% high concentration presterilized and filtered NaCl solution (diluted by pretreated coelomic fluid) or DDW (double distilled water). Transfer the sea cucumber coelomic fluid into a sterile bottle, seal the bottle, and store the fluid at 4 &#176;C for further experiments.</li>
	</ol>
	</li>
	<li>Leibovitz&#39;s L-15 cell culture medium optimization
	<ol>
		<li>Weigh 5.05 g of NaCl, 0.135 g of KCl, 0.15 g of CaCl2, 0.25 g of Na2SO4, 0.975 g of MgCl2, 0.25 g of glucose, and 6.25 mg of taurine and dilute them in 40 mL of Leibovitz&#39;s L-15 medium in a 50 mL sterile centrifuge tube. Agitate the tube on a shaker for 1 h to ensure the salts have dissolved completely.</li>
		<li>Add 2.5 mL of L-glutamine (100 mg/mL) and 500 &#956;L of VE solution (1.75 mg/L) into previously prepared Leibovitz&#39;s L-15 medium and further filter the medium through 0.22 &#956;m membrane filters.</li>
		<li>Adjust the total medium volume to 500 mL with fresh Leibovitz&#39;s L-15 medium and with 100 mL of previously prepared coelomic fluid; next, adjust the pH to 7.6 using NaOH solution. Keep the operation process in a sterile environment. The compounding ratio of coelomic fluid can be 10%&#8211;50%, and 20% is sufficient for A. japonicus intestinal cells culture.</li>
	</ol>
	</li>
</ol>
2. Intestinal Cell Preparation
<ol>
	<li>Sea cucumber intestine processing
	<ol>
		<li>Anaesthetize healthy sea cucumbers in an ice box. Dissect and collect the anterior intestines, then section the tissue samples vertically and remove the inner contents.</li>
		<li>Wash the tissue samples in phosphate buffered saline (PBS) twice and disinfect them by immersion in an aqueous ethanol solution (75% by volume) for no longer than 2 s.</li>
		<li>Wash the tissue samples in PBS three times to remove ethanol and transfer about 100 mg of tissue sample into a 2.0 mL sterile microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Cell collection
	<ol>
		<li>Add 1.5 mL of the pre-optimized culture medium to the sea cucumber intestinal tissue block and mince the block with sterilized surgical scissors until the solution is cloudy. 
		NOTE: For optional simplified protocol, add 0.5 mL of pre-optimized culture medium to the sea cucumber intestinal tissue block and cut the block with sterilized surgical scissors into 1 mm3. Directly transfer the samples to culture dishes followed by subsequent incubating steps.</li>
		<li>Add 400 &#956;L of trypsin (0.25%), mix the solution by inversion, and incubate it for 5 min at room temperature; then, filter the solution using a 100 &#956;m cell strainer. 
		NOTE: It is optional to add trypsin for cell dispersion when treating different tissue samples. Ethylenediaminetetraacetic acid (EDTA) should be contained in trypsin solution to reduce the inhibitory activity from Ca2+ and Mg2+ in culture medium.</li>
		<li>Collect the filtrate to a new sterile 2.0 mL microcentrifuge tube, centrifuge at 1,700 x g for 3 min, discard the supernatant, then resuspend the pellet in culture medium (supplemented with antibiotics) and wash it twice. 
		NOTE: Prepare fresh pre-optimized culture medium supplemented with 2% penicillin-streptomycin solution (10,000 U/mL penicillin and 10 mg/mL streptomycin) and 1% gentamicin (4 mg/mL) before the beginning of the experiment.</li>
	</ol>
	</li>
</ol>
3. Cell Culture
<ol>
	<li>Incubator presetting: Preset the incubator for cell culture and run it in advance for at least 24 h with temperature of 18 &#176;C and saturated humidity. 
	NOTE: Feed CO2 into the incubator depending on the cell culture medium properties; no CO2 needs to be supplied when using the basic medium of Leibovitz&#39;s L-15.</li>
	<li>First stage cell culture
	<ol>
		<li>To inhibit the growth of microbes and to promote the proliferation during the initial stage of cell culture, add 10 mL of penicillin-streptomycin solution (10,000 U/mL penicillin and 10 mg/mL streptomycin) and 0.5 mL of gentamicin (40 mg/mL) into every 500 mL of pre-optimized culture medium. Furthermore, supplement every 500 mL culture medium with 0.6 mL of insulin (10 mg/mL), 100 &#956;L of insulin-like growth factor (0.1 &#956;g/&#956;L), and 25 &#956;L of fibroblast growth factor (0.1 &#956;g/&#956;L).</li>
		<li>Collect the cells into 1.5 mL tubes, resuspend them using 200 &#956;L of indicated medium, and pipet them into &#966; 4 cm dishes.</li>
		<li>Culture the cells in an incubator and add 2.0 mL of indicated medium to the cell culturing dishes after 6 h. Change half of the medium every 12 h until reaching the next stage. 
		NOTE: Handle the medium change gently, because the cells are not attached to the dishes tightly. Poly-D-lysine-coated dishes can be used for loosely attaching to the dish cells.</li>
	</ol>
	</li>
	<li>Second stage cell culture
	<ol>
		<li>To reduce the adverse effects of antibiotics to the cultured cells, reduce the concentration of indicated antibiotics (penicillin, streptomycin, and gentamicin) in the culture medium by half. 
		NOTE: The usage of insulin and growth factors depends on the cell culture conditions and is optional.</li>
		<li>Replace the cell culture medium; conduct medium changes every two to three days depending on the cell density. 
		NOTE: Observe the cultured cells daily under a microscope and record the growth conditions.</li>
	</ol>
	</li>
	<li>Cell passaging 
	NOTE: Passage and subculture the cells, when the primary cell density reaches 60%.
	<ol>
		<li>Wash the cultured cells twice using PBS at room temperature. Add 200 &#956;L of trypsin solution (0.25%) to each dish and manually agitate the dish ensuring the whole bottom is covered. Discard the trypsin solution and incubate the cells for 5 min at room temperature.</li>
		<li>Wash the cells with 1.0 mL of fresh culture medium by pipetting and resuspending the cells. Transfer 0.5 mL of cell suspension to a new dish, add 1.5 mL of fresh medium, and incubate the cells at 18 &#176;C. 
		NOTE: Cell scrapers can be used for cell collection when the trypsin solution fails to digest and detach cells from the dishes (some cell lines are too adhesive). However, do not conduct both methods simultaneously.</li>
		<li>Change the medium after 12 h and observe the cells under a microscope to evaluate the conditions. Culture the cells for further experimental assays.</li>
	</ol>
	</li>
</ol>
4. Apoptosis Induction and Detection in A. japonicus Intestinal Cells
<ol>
	<li>Cell culture and dexamethasone treatment
	<ol>
		<li>Prepare intestinal cells following the previously introduced protocol and add the cells dropwise to a 12-well plate at a cell volume of 2 x 106 per well.</li>
		<li>After three days in culture, following the steps of the &#34;first stage cell culture&#34;, wash the cells three times with PBS and replace the medium with optimized medium (without antibiotics and growth factors).</li>
		<li>Dilute dexamethasone (DXMS) in culture medium to prepare fresh 2 &#956;M and 200 &#956;M DXMS solutions before beginning the experiments.</li>
		<li>Add DXMS solutions in different concentrations to cultured cells grown with the same volume of culturing medium; set three experimental groups including control (CTL), 1 &#956;M, and 100 &#956;M DXMS.</li>
	</ol>
	</li>
	<li>Hoechst staining 
	<ol>
		<li>Wash the cells with PBS three times after incubation with/without DXMS for the indicated time periods (0 h, 24 h, and 48 h).</li>
		<li>Add 300 &#956;L of Hoechst 33258 solution per well to a 12-well plate and incubate at 18 &#176;C for 30 min. Gently agitate the plate to cover all cells ensuring their staining.</li>
		<li>Remove the Hoechst staining solution and fix the cells by adding 300 &#956;L of a 4% paraformaldehyde solution (in PBS) to each well. Gently agitate for 15 min. 
		CAUTION: Paraformaldehyde is moderately toxic by skin contact or inhalation, and it is designated as a probable human carcinogen. Chemical fume hoods, vented balance enclosures, or other protective measures should be used during the weighing and handling of paraformaldehyde.</li>
		<li>Wash the fixed cells three times in PBS. Do not discard the PBS after washing to keep cells covered.</li>
	</ol>
	</li>
	<li>Fluorescent microscopy analysis
	<ol>
		<li>Turn on the fluorescent microscope hardware including the mercury lamp power, fluorescent light power, and PC. Log into the operating system account, launch the software, and check its configuration.</li>
		<li>Place the prepared plate on the microscope stage. Position the sample over the objective lens using the stage controller.</li>
		<li>Find the cells of interest under the light microscope, switch to fluorescent microscopy, and capture the images by tuning the parameters. 
		NOTE: To observe the Hoechst 33258 fluorescent signal bound to nuclei DNA, fluorescence microscopy should be conducted with excitation and emission at approximately 352 nm and 461 nm, respectively.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

Extensive research efforts have been devoted to establishing cell lines in last decades, however, it is still difficult to make a progress on long-term culture of cells from marine invertebrates14,22. It has been reported that cultured cells from regenerating holothurian tissues were viable for a long period of time and high activity of proliferation can be detected in specific cells17,23. However, for th...

Culturing cells under artificially controlled conditions, and not in their natural environment, provides uniform experimental materials for biological studies, especially for species which cannot be easily cultured in a laboratory environment. Marine invertebrates account for more than 30% of all animal species1, and they provide numerous biological materials for undertaking research on the regulatory mechanisms of specific biological processes, such as regeneration2,3, stress response4, and environmental adaptation5,6.
The sea cucumber, Apostichopus japonicus, is one of the most studied echinoderm species inhabiting temperate waters along the North Pacific coast. It is well known as a commercially important species and maricultured on a large scale in East Asia, especially in China7. Numerous scientific questions regarding A. japonicus, including the regulatory mechanisms underlying intestinal regeneration after evisceration8 and degeneration in aestivation9, metabolic control10,11, and immune response12,13 under thermal or pathogenic stresses, have attracted the attention of researchers. However, compared with well-studied model animals, basic research, especially on the cellular level, is limited by technical bottlenecks, such as the lack of advanced cell culture methods.
Researchers have devoted much effort to establishing cell lines, but they have also faced many challenges and no cell line from any marine invertebrate has been established yet14. However, primary cell cultures from marine invertebrates have advanced in last decades15,16, and they have provided an opportunity for experimentation on the cellular level. For example, the regenerating intesine from A. japonicus has been utilized as a source of cells for long-term cell cultures which provided a practical method for primary cell culture of marine invertebrates17. This protocol combined and optimized invertebrate cell culture approaches and developed a widely suitable primary culture method for sea cucumber or other marine invertebrates.
Apoptosis is an intrinsic cell suicide program triggered by various exogenous and endogenous stimuli. Coordinated apoptosis is crucial to many biological systems18,19, and it has been implicated in the intestinal regression of sea cucumber during aestivation9. To investigate the apoptotic process in organisms of interest, a series of methods, including Hoechst staining and microscopy assays, have been established and successfully applied20. Here, we conducted apoptosis induction and detection in primary cultured intestinal cells of sea cucumber to assess the usability of primary cells in biological studies of marine invertebrates. Dexamethasone, one of the commonly used synthetic glucocorticosteroids21, was used to induce apoptosis in cultured intestinal cells from sea cucumber, and significant Hoechst 33258 signal was successfully detected in the stained cells by fluorescent microscopy.

<table><tbody><tr><td>0.1 &amp;mu;m filter</td><td>Millipore</td><td>SLVV033RS</td><td></td></tr><tr><td>0.22 &amp;mu;m filter</td><td>Millipore</td><td>SLGP033RB</td><td></td></tr><tr><td>0.25% Trypsin</td><td>Genom</td><td>GNM25200</td><td></td></tr><tr><td>100 &amp;mu;m filter</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>4 cm dishes</td><td>ExCell Bio</td><td>CS016-0124</td><td></td></tr><tr><td>4% paraformaldehyde solution</td><td>Sinopharm Chemical Reagent</td><td>80096618</td><td>in PBS</td></tr><tr><td>Benchtop Centrifuges</td><td>Beckman</td><td>Allegra X-30R</td><td></td></tr><tr><td>BeyoClick EdU-488 kit</td><td>Beyotime</td><td>C0071S</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent</td><td>10005817</td><td></td></tr><tr><td>Constant temperature incubator</td><td>Lucky Riptile</td><td>HN-3</td><td></td></tr><tr><td>Dexamethasone</td><td>Sinopharm Chemical Reagent</td><td>XW00500221</td><td></td></tr><tr><td>Electric thermostatic water bath</td><td>senxin17</td><td>DK-S28</td><td></td></tr><tr><td>Ethanol</td><td>Sinopharm Chemical Reagent</td><td>80176961</td><td>75%</td></tr><tr><td>Fibroblast Growth Factor(FGF)</td><td>PEPROTECH</td><td>100-18B</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Leica DMI3000B</td><td>DMI3000B</td><td></td></tr><tr><td>Garamycin</td><td>Sinopharm Chemical Reagent</td><td>XW14054101</td><td></td></tr><tr><td>Glucose</td><td>Sinopharm Chemical Reagent</td><td>63005518</td><td></td></tr><tr><td>Hoechst33258 Staining solution</td><td>Beyotime</td><td>C1017</td><td></td></tr><tr><td>Insulin</td><td>Sinopharm Chemical Reagent</td><td>XW1106168001</td><td></td></tr><tr><td>Insulin like Growth Factor(IGF)</td><td>PEPROTECH</td><td>100-11</td><td></td></tr><tr><td>KCl</td><td>Sinopharm Chemical Reagent</td><td>10016308</td><td></td></tr><tr><td>Leibovitz&#039;s L-15</td><td>Genom</td><td>GNM41300</td><td></td></tr><tr><td>L-glutamine (100 mg/mL)</td><td>Genom</td><td>GNM-21051</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent</td><td>XW77863031</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent</td><td>10020518</td><td></td></tr><tr><td>NaCl</td><td>Sinopharm Chemical Reagent</td><td>10019308</td><td></td></tr><tr><td>NaOH</td><td>Sinopharm Chemical Reagent</td><td>10019718</td><td></td></tr><tr><td>PBS</td><td>Solarbio</td><td>P1020</td><td>pH7.2-7.4</td></tr><tr><td>Penicillin-Streptomycin</td><td>Genom</td><td>GNM15140</td><td></td></tr><tr><td>PH meter</td><td>Bante</td><td>A120</td><td></td></tr><tr><td>Taurine</td><td>SIGMA</td><td>T0625</td><td></td></tr><tr><td>VE</td><td>Seebio</td><td>185791</td><td></td></tr></tbody></table>

apoptosis induction and detection in a primary culture of sea cucumber intestinal cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

Here, we established primary intestinal cell culture of A. japonicus and passaged the cells. Figure 1 shows round cells in different stages of culturing. And the EdU staining assays provide direct evidences to reveal the proliferative activity of these round cells in later stage (Figure 2). We also slightly adjusted the protocol, culturing minced tissue blocks instead of filtrated cells; furthermore, a spindle cell type could be cultured successfully. T...

Watch this Scientific Journal Video about Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells at JoVE.com

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

apoptosis-induction-detection-primary-culture-sea-cucumber-intestinal

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

6.9K Views.  Zhejiang Ocean University. This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Video: Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Cancer Research

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Biochemistry

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.

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

Immunology and Infection

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...