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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 &#956;m filter</td>
			<td>Millipore</td>
			<td>SLVV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;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 &#956;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>CaCl2</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&#39;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>MgCl2</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>XW77863031</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2SO4</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

Research

JoVE Journal

Biology

6.8K 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

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Methods Article

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