Name: identification of intracellular signaling events induced in viable cells by interaction with neighboring cells undergoing apoptotic cell death
Uploaded: 2016-12-27T13:00:00
Description: Watch this Scientific Journal Video about Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death at JoVE.com

This work was supported, in whole or in part, by institutional funds from Dr. Jose&#769; A. Arruda and the Section of Nephrology, University of Illinois at Chicago (to J. S. L.); and funding from the Department of Medicine of McGill University and the Research Institute of the McGill University Health Centre (to J. R.)

1. Preparations
Note: In this protocol, two or more cell lines, or primary cell cultures, are independently prepared under specific conditions. These preparations include apoptotic cells (Protocol 1), necrotic cells (Protocol 2), and healthy responder cells (Protocol 3). After independent preparation, apoptotic or necrotic cells are added to responder cells and the resulting signal transduction is monitored (Protocols 4, 5, and 6).
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	<li>Prepare culture media and reagents
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		<li>Pre...

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We provide here a protocol for characterizing the intracellular signaling events induced in viable cells following their exposure to cells undergoing apoptosis, or other forms of cell death. The protocol emphasizes the multiple mechanisms by which exposure to apoptotic cells can modulate intracellular signaling pathways within viable responder cells. These mechanisms include: the interaction (via bridging molecules or surface determinants on the dead cell or its shed vesicles and microvesicles, often termed apoptotic bod...

Apoptosis, or regulated cell death1, contributes in an essential manner to the maintenance and development of tissues. Viewed most simply, apoptosis permits aged, damaged, or excess cells to be eliminated without harm to surrounding tissues2,3. The contribution of apoptosis to tissue homeostasis, however, is considerably more dynamic and varied. Cells dying by apoptosis acquire multiple new activities, both secreted and cell-associated, which enable them to influence the function of adjacent live cells4-10. Earlier studies focused on the ability of apoptotic cells to suppress inflammation11-16, but apoptotic cells also modulate a broad range of cellular functions, including such vital activities as survival4,9,10, proliferation4,9,10, differentiation17, migration18, and growth19. Moreover, these effects are not restricted to professional phagocytes, like macrophages, but extend to virtually all cell types and lineages, including traditionally non-phagocytic cells, such as epithelial and endothelial cells7,9,10,18-21.
The specific response of adjacent live cells following exposure to apoptotic cells depends on multiple factors that relate to both the viable responding cells and the apoptotic cells themselves. For example, although exposure to apoptotic cells inhibits the proliferation of both murine macrophages and kidney proximal tubular epithelial cells (PTECs), these two cell types differ dramatically in their survival response to apoptotic cells4,6,9,10. Apoptotic cells promote the survival of macrophages, but induce the apoptotic death of PTECs4,6,9,10. Notably, the response to apoptotic cells may differ even among responding cells of the same lineage, depending on the responding cell&#39;s organ of origin10 (e.g., kidney PTECs versus mammary epithelial cells) or state of activation22 (e.g., neutrophils). Conversely, apoptotic cells may evoke different responses in the very same cell depending on the nature of the apoptotic stimulus10,23 or the stage of apoptosis10,14.
Given the diverseness and complexity of the response to apoptotic cells, great care must be taken in dissecting the signaling events and pathways responsible for any particular outcome. First, responses requiring direct physical interaction between viable and apoptotic cells must be differentiated from those elicited by soluble mediators released or generated by the apoptotic cell3,6-10. If physical interaction is required, then a further differentiation should be performed. Signaling events may depend on receptor-mediated recognition of the apoptotic cell, independent of subsequent engulfment, or on phagocytic uptake, independent of the specific receptor that binds the apoptotic cell3-7,9,10,19. In the latter instance, the response is not specific to apoptotic cells, and can be triggered by any phagocytic material4,6.
The importance of these distinctions can again be appreciated by contrasting the responses of macrophages and kidney PTECs. For both cell types, exposure to apoptotic cells alters the activity of the pro-survival kinase Akt. Modulation is dependent on physical interaction, since separation of responding and apoptotic cells by a 0.4 &#956;m polycarbonate membrane abolishes the response4,9,10,19. However, the response of PTECs is receptor-mediated and independent of phagocytosis, whereas the response of macrophages is driven by phagocytosis4,9,10,19. This conclusion is reinforced by the fact that exposure to latex beads, a neutral phagocytic stimulus, has no effect on Akt activity in PTECs, but mimics the effect of apoptotic cells in macrophages4,9.
Although less well studied, cells dying by necrosis, or accidental cell death1, also modulate the function of nearby viable cells3-10,19. Like apoptotic cells, necrotic cells exert their effects through a variety of mechanisms, particularly the leakage of intracellular contents through their ruptured cell membrane3,5,6,9,24. Many cells, including PTECs and macrophages, possess distinct non-competing receptors for cells dying by necrosis5,9. Engagement of these receptors induces signaling events that are often opposite to those induced by engagement of the receptors for apoptotic cells4-10,19. For example, in PTECs, necrotic cells increase phosphorylation of Akt, whereas apoptotic cells decrease phosphorylation9,10,19.
Here, we describe a protocol for identifying intracellular signaling events induced in viable kidney PTECs through receptor-mediated recognition of adjacent apoptotic PTECs9,10,19. While the protocol is specific for a conditionally immortalized PTEC cell line known as BU.MPT cells9,10,19,25,26, it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. Importantly, the use of apoptotic cells as a cell stimulus poses certain inherent experimental difficulties not present with soluble ligands. The most important of these is that apoptotic cells must be added as a suspension rather than as a solution. These difficulties, as well as strategies to minimize or circumvent them, are discussed within the protocol. Nearly all of the techniques described in the protocol are straightforward and standard to cell culture. The advantage of this protocol lies in its attention to the multiple mechanisms by which apoptotic cells modulate signal transduction within responding cells. These mechanisms include the binding via surface determinants or bridging molecules to specific receptors on the responding cell, the release of soluble mediators, and/or the engagement of the phagocytic machinery. Necrotic cells are included in all experiments to ensure that results are specific to the mode of cell death, and not a generalized response to dead cells. A careful approach, as recommended in this protocol, is critical to our understanding of the ever expanding influence that dying cells exert on their live neighbors, in both health and disease.

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			<td height="21" style="height:21px;width:461px;">Cell culture materials</td>
			<td style="width:263px;"></td>
			<td style="width:127px;"></td>
			<td style="width:427px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, 1X with [4.5 g/L] glucose, L-glutamine &#38; sodium-pyruvate</td>
			<td>Mediatech (Manassas, VA)</td>
			<td style="width:127px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone Fetal Clone II, fetal bovine serum</td>
			<td style="width:263px;">GE Healthcare (Logan, UT)</td>
			<td style="width:127px;">SH30066.03</td>
			<td>add 10% (v/v)&#160; to the culture medium A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin-Glutamine, 100X</td>
			<td style="width:263px;">Life Technologies (Grand Island, NY)</td>
			<td style="width:127px;">10378-016</td>
			<td>add 100 units/mL to the culture media A and B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#947;-Interferon, mouse recombinant, from Escherichia coli</td>
			<td style="width:263px;">Calbiochem, Millipore (Billerica, MA)</td>
			<td style="width:127px;">407303</td>
			<td>add 10 units/mL to the culture medium A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Falcon polystyrene tissue-culture treated dishes, non-pyrogenic</td>
			<td style="width:263px;">Corning (Durham, NC)</td>
			<td style="width:127px;">353003</td>
			<td>sterile, 100 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Falcon polystyrene centrifuge tubes, conical bottom</td>
			<td style="width:263px;">Corning (Durham, NC)</td>
			<td style="width:127px;">352095</td>
			<td>sterile, 15 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Trypsin-EDTA, 0.05%, Gibco</td>
			<td style="width:263px;">Fisher (Waltham, MA)</td>
			<td style="width:127px;">25300062</td>
			<td>sterile, 100 mL</td>
		</tr>
		<tr height="21">
			<td>Name</td>
			<td>Company</td>
			<td>Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemicals and inhibitors</td>
			<td style="width:263px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Staurosporine, from Streptomyces sp.</td>
			<td style="width:263px;">Sigma (St. Louis, MO)</td>
			<td style="width:127px;">S4400</td>
			<td>use at [1 &#956;g/mL] for 3 h to induce apoptosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Epidermal growth factor (EGF), [1 mg/ml]</td>
			<td style="width:263px;">Millipore (Billerica, MA)</td>
			<td style="width:127px;">EA140</td>
			<td>use at 50 nM to stimulate responder cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">UltraPure, 0.5 M EDTA, pH 8.0</td>
			<td style="width:263px;">Life Technologies (Grand Island, NY)</td>
			<td style="width:127px;">15575-038</td>
			<td>use 5 mM to detach adherent cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Trypan blue solution, 0.4%</td>
			<td style="width:263px;">MP Biomedicals (Santa Ana, CA)</td>
			<td style="width:127px;">91691049</td>
			<td>for dead cells staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Paraformaldehyde Solution, 4% in DPBS</td>
			<td style="width:263px;">Affymetrix (Santa Clara, CA)</td>
			<td>19943</td>
			<td>use 0.4% (v/v) in 1&#215; DPBS for cell fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Dulbecco&#39;s phosphate-buffered saline (DPBS), 1&#215;, Gibco</td>
			<td style="width:263px;">Life Technologies (Grand Island, NY)</td>
			<td>14040133</td>
			<td>stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:461px;">Dulbecco&#39;s phosphate-buffered saline (DPBS), Ca+2- and Mg+2-free, 1&#215;, Gibco</td>
			<td style="width:263px;">Life Technologies (Grand Island, NY)</td>
			<td style="width:127px;">14190367</td>
			<td>stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:461px;">Cytochalasin D, 4 mM in dimethyl sulfoxide (DMSO), from Zygosporium mansonii</td>
			<td style="width:263px;">Sigma (St. Louis, MO)</td>
			<td style="width:127px;">C8273</td>
			<td>use at [4 &#956;g/mL] to inhibit phagocytosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:263px;">Sigma (St. Louis, MO)</td>
			<td style="width:127px;">D2650</td>
			<td>solvent for cytochalasin D</td>
		</tr>
		<tr height="21">
			<td>Name</td>
			<td>Company</td>
			<td>Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell lysis buffer ingredients</td>
			<td style="width:263px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Tris-buffered saline (TBS), 10&#215;, pH 7.4</td>
			<td style="width:263px;">BioRad (Hercules, CA)</td>
			<td style="width:127px;">1706435</td>
			<td>for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium pyrophosphate</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">221368</td>
			<td>10 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium deoxycholate</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">D6750</td>
			<td>0.5% w/v for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">L3771&#160;</td>
			<td>0.1% w/v for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">G5516</td>
			<td>10% for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium fluoride</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">450022</td>
			<td>25 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Complete mini EDTA-free protease inhibitor cocktail tablet</td>
			<td style="width:263px;">Roche Diagnostics (Indianapolis, IN)</td>
			<td style="width:127px;">4693159001</td>
			<td>for cell lysis buffer, 1 tablet per 10 mL of buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Triton X-100</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">T8787</td>
			<td>10% for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Dithiothreitol (DTT)</td>
			<td style="width:263px;">MP Biomedicals (Santa Ana, CA)</td>
			<td style="width:127px;">11DTT00005</td>
			<td>1 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td style="width:263px;">MP Biomedicals (Santa Ana, CA)</td>
			<td style="width:127px;">4800263</td>
			<td>1 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sodium orthovandate</td>
			<td style="width:263px;">MP Biomedicals (Santa Ana, CA)</td>
			<td>2159664</td>
			<td>10 mM for cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td>Name</td>
			<td>Company</td>
			<td>Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:263px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sorvall T6000B centrifuge</td>
			<td style="width:263px;">Du Pont (Wilmington, DE)</td>
			<td style="width:127px;">T6000B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Fisher Tissuemat, water bath</td>
			<td style="width:263px;">Fisher (Waltham, MA)</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Sonic Dismembrator (sonicator), model 100</td>
			<td style="width:263px;">Fisher (Waltham, MA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Miscellaneous</td>
			<td style="width:263px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Cell counting chamber, Neubauer</td>
			<td style="width:263px;">Hawksley (Lancing, Sussex, UK)</td>
			<td style="width:127px;">AC2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Annexin V-FITC Apoptosis Detection Kit II</td>
			<td style="width:263px;">Millipore (Billerica, MA)</td>
			<td style="width:127px;">CBA059</td>
			<td>for PI and Annexin V staining to determine the stage of apoptosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Eppendorf microcntrifuge tubes, 1.5 ml</td>
			<td style="width:263px;">Sigma-Aldrich (St. Louis, MO)</td>
			<td style="width:127px;">T9661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">Transwell permeable support, 0.4 &#956;m polycarbonate membrane</td>
			<td style="width:263px;">Corning (Corning, NY)</td>
			<td style="width:127px;">3413</td>
			<td>sterile, polystyrene, tissue culture treated</td>
		</tr>
		<tr height="21">
			<td>Name</td>
			<td>Company</td>
			<td>Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibodies</td>
			<td style="width:263px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:461px;">Phospho-glycogen synthase kinase-3&#945;/&#946; (GSK-3&#945;/&#946;) (Ser21/9), polyclonal antibody</td>
			<td style="width:263px;">Cell Signaling Technology</td>
			<td style="width:127px;">9331</td>
			<td>rabbit polyclonal antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:461px;">Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antobody ( (14C10) )</td>
			<td style="width:263px;">Cell Signaling Technology</td>
			<td style="width:127px;">2118</td>
			<td>rabbit monoclonal antibody</td>
		</tr>
	</tbody>
</table>

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.

In Figure 1, we provide a schematic depicting the timing and critical steps involved in the preparation of apoptotic cells (Protocol 1) and necrotic cells (Protocol 2) and the stimulation of responder cells with suspensions of apoptotic and necrotic cells (Protocol 4).
In Figure 2, we provide representative results showing the effect of apoptotic cells on phosphorylation of the two isoforms of t...

Watch this Scientific Journal Video about Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death at JoVE.com

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

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 ...

The overall goal of this procedure is to identify signaling events induced and viable responder cells following their physical interaction with nearby dead cells. This method can help answer key questions in the fields of cell biology, pathology and physiology such as the influence that dead or dying cells exert on their live neighbors. The main advantages of this technique are that it is straightforward and inexpensive, and that it can be applied to study biologic effects induced in live cells following their interaction with neighboring cells undergoing apoptosis.Demonstrating the technique will be Lanfei Feng, a technician in my laboratory. To begin this procedure after passing the BUNPT cells on to 100 millimeter vacuum gas plasma treated tissue culture dishes, grow them in culture medium A to confluence and humidified 5%carbon-dioxide atmosphere at 37 degrees Celsius. After that, rinse the adherent cell monolayer three times with culture medium B.Then, induce apoptosis by incubating the cells in culture medium B, containing staurosporine, a nonselective protein kinase inhibitor, for 3 hours at 37 degrees Celsius.After 3 hours, collect the medium containing floating apoptotic cells that have detached from the monolayer. Subsequently, centrifuge this medium for ten minutes at 500 times G.Afterward, discard the supernatant, wash the pellet three times with culture medium B, before adding it back to the adherent cells. Following this washing step, detach the remaining adherent cells by adding 5 millimolar EDTA in 1X calcium and magnesium free DPBS.Collect the medium containing detached apoptotic cells and put the detached cells with the floating apoptotic cells in a sterile 15 milliliter polystyrene centrifuge tube. Then wash the apoptotic cells three times by centrifugation for ten minutes at 500 x G.Followed by resuspension in ten milliliters of 1X calcium and magnesium-free DPBS per wash. After the last wash, suspend the apoptotic cells in fresh culture medium B at 5x10^6 cells per milliliter, before using them to stimulate the responder cells.Alternatively, fix the washed apoptotic cells in 0.4 paraformaldehyde in 1X DPBS for 30 minutes, then wash them three times with culture medium B, and suspend them in it at 5x10^6 cells per milliliter before using them to stimulate the responder cells. After passaging the BUNPT cells on to 100 millimeter diameter sterile polystyrene vacuum gas plasma treated tissue culture dishes, grow them to confluence and humidified 5%carbon-dioxide atmosphere under permissive conditions. Then rinse the adherent cell monolayer three times with 10 mLs of culture medium B per rinse.Subsequently, detach the cells by adding 5 mM EDTA in 1X calcium and magnesium-free DPBS. Collect the detached cells from the EDTA containing medium and add the cell suspension to a sterile 15 mL polystyrene centrifuge tube. Next, wash the cells three times by centrifugation for ten minutes at 500 times G and resuspension in 10 mLs of calcium and magnesium-free DPBS per wash.After the last wash, suspend the cells in fresh culture medium B at 5x10^6 cells per milliliter. Following this, induce necrosis by heating the cells to 70 degrees Celsius for 45 minutes in a water bath, gently vortexing this cell suspension every ten minutes. After the induction of necrosis, incubate the cells for two hours in a humidified 5%carbon-dioxide atmosphere at 37 degrees Celsius, and gently vortex the cell suspension every 15 minutes.In this procedure, grow the BUNPT cells in sterile 100 millimeter tissue culture dishes under permissive conditions, until they achieve about 85%confluence. Afterward, aspirate culture medium A and rinse the cells three times with 10 mL of culture medium B per rinse. Then, serum starve the cells in culture medium B for 18 to 24 hours in a humidified 5%carbon-dioxide atmosphere under non-permissive conditions, in order to induce quiescence.Label a separate tissue culture dish of confluent BUNPT cells for the six experimental conditions for the next procedure. Now, aspirate culture medium B from the pre-labeled dishes of quiescent BUNPT responder cells. To prepare the 100 millimeter dishes in different conditions, add two mL of either culture medium B with no cells, the apoptotic cell suspension at 5X10^6 cells per milliliter in culture medium B, or the necrotic cell suspension at 5X10^6 cells per mL in culture medium B.Achieve a dead to responder cell ratio of one to one by adding to mL of dead cells at 5X10^6 cells per milliliter to a confluent 100 millimeter dish, which contains 10X10^6 cells.Gently swirl the dishes. Then incubate them at 37 degrees Celsius in the humidified 5%carbon-dioxide atmosphere. After 30 minutes, stimulate the responder cells with either vehicle or 15 nMOL EGF, according to the pre-labeling of the culture dish.Incubate the cells for 15 minutes at 37 degrees Celsius in a humidified 5%carbon-dioxide atmosphere. Afterward, wash away the dead cells by adding five millimeters of ice-cold DPBS. Swirl the dish and aspirate the wash fluid.Repeat the wash three times. Then immediately place the responder cell dishes on ice for further processing. This figure shows that exposure of quiescent BUNPT responders to apoptotic cells for 30 minutes strongly inhibited basal phosphorylation of GSK3 alpha beta.To prevent physical interaction between BUNPT responders and apoptotic cells, BUNPT responders were grown in a permeable support system, and separated from apoptotic targets by of 0.4 micron polycarbonate membrane. Prevention of physical interaction between BUNPT responders and apoptotic cells abolished the ability of apoptotic targets to inhibit phosphorylation of GSK3 alpha beta. To evaluate the role of phagocytosis, the cytoskeletal inhibitor Cytochalasin D was used to prevent phagocytic uptake of dead cells.Inhibition of phosphorylation of GSK3 alpha beta in response to apoptotic cells occurred in the absence of phagocytosis. Once mastered, this technique can be done in 4-6 hours if it is performed properly, excluding the time required for gel electrophoresis and immunoblotting. While attempting this procedure, it's important to remember that using a cellular suspension as opposed to a soluble ligand as a stimulus poses several inherent and largely unavoidable experimental obstacles.Using the same procedure, one can study other cell functions, such as survival or proliferation or cell growth in order to determine the functional consequences of the signaling events that are induced in live responding cells following their interaction with nearby dead cells. After watching this video, you should have a good understanding of how to identify specific signaling events that are induced in viable responder cells following their physical interaction with nearby dead cells.

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SCIENTISTS IN ACTION

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...