This work was funded by a Natural Sciences and Engineering Research Council (NSERC) Canada grant to MGM. JR is a NSERC-Vanier student fellow.

N.B.: Each of the following steps should be performed under sterile conditions in a laminar flow hood as required for mammalian cell culture. In addition, the general guidelines for optimal sterile cell cultivation apply, e.g., discarding tips any time they may lead to cross-contamination, reducing the amount of time cells are exposed to the air when performing entire media changes, properly but gently stirring all cell suspensions to ensure their homogenous pipetting, etc. Moreover, inserts are a kind of plasticware that require special handling. First, whenever inserts are manipulated, avoid touching the fragile membrane, which tears easily and could therefore jeopardize the experiment. Also, it is not suitable to perform vacuum aspiration of the cell culture medium, as there is a risk of perforating the membrane or dissociating adherent cells. Next, inserts hang loosely in the multiwell tissue culture plate and, thus, caution must be employed when moving the plasticware or when pipetting to avoid dissociating adherent cells. In addition, when using inserts with large pore sizes, there is a possibility that the cell culture medium seeps through the membrane and, therefore, it is important to frequently monitor the level of liquid. Finally, note that the following protocol is designed for adherent cells and requires minor modifications in order to be suitable for suspension cells.
1. General guidelines for conducting insert co-culture experiments
<ol>
	<li>Seeding cell type #1 in inserts
	<ol>
		<li>Unwrap the inserts from their packaging.</li>
		<li>Place the inserts in an empty multiwell tissue culture plate of the proper dimension. To do so, grip the uppermost edge of the insert using tweezers.</li>
		<li>To improve the attachment and spreading of adherent cells, condition the inserts with cell culture medium prior to seeding. To do so, cover the entire surface of the membrane with cell culture medium using a micropipette. 
		NOTE: Do this for as many inserts as required.</li>
		<li>Replace the lid on the plate and incubate for at least 1 hr or O/N under the same conditions (usually 37&#176;C, 5-10% CO2).</li>
		<li>When the inserts are conditioned, remove all of the cell culture medium using a micropipette. Discard the used medium.</li>
		<li>Seed cell type #1 in fresh cell culture medium in the same manner as in a multiwell plate. To do so, draw an appropriate volume of the cell suspension with a micropipette and dispense the liquid in the insert. 
		NOTE: Prepare as many inserts as required.</li>
		<li>After seeding all the inserts, gently rock the plate left and right, then back and forth in order to distribute the cells evenly. Avoid making circular motions, as this will cause the cells to accumulate in the center of the inserts.</li>
		<li>Place the lid on the plate and incubate as specified by as per cellular requirements (usually 37&#176;C, 5-10% CO2).</li>
	</ol>
	</li>
	<li>Seeding cell type #2 in multiwell tissue culture plates
	<ol>
		<li>Seed cell type #2 in fresh cell culture medium according to Section 1.</li>
		<li>Prepare as many wells as required for the number of inserts. Rock the plate as in step 1.1.7) to ensure that the cells are evenly distributed in the wells.</li>
		<li>Place the lid on the plate and incubate under the same conditions (usually 37&#176;C, 5-10% CO2).</li>
	</ol>
	</li>
	<li>Refreshing medium in inserts
	<ol>
		<li>Using a micropipette, remove part or all of the cell culture medium, in the inserts containing cell type #1. Discard the used medium.</li>
		<li>Draw an appropriate volume of fresh cell culture medium. Gently rest the tip on the inner wall of the insert and slowly dispense the cell culture medium.</li>
		<li>Place the lid on the plate and incubate as per step 1.1.4.</li>
	</ol>
	</li>
	<li>Refreshing medium in multiwell tissue culture plates
	<ol>
		<li>Using a micropipette, remove part or all of the cell culture medium in the wells containing cell type #2. Discard the used medium.</li>
		<li>Draw an appropriate volume of fresh cell culture medium. Rest the tip on the inner wall of the well and slowly dispense the cell culture medium.</li>
		<li>Place the lid on the plate and incubate according to previously established cell culture protocols.</li>
	</ol>
	</li>
	<li>Transferring inserts containing cell type #1 to multiwell tissue culture plates containing cell type #2. 
	NOTE: Perform this step when both cell types have reached the appropriate growth stage.
	<ol>
		<li>Prior to transferring the inserts into wells, make any necessary medium changes, as previously described in steps 1.3) and 1.4). 
		NOTE: At this point, it is important to dispense the appropriate volumes of media in both compartments as specified by the insert manufacturer&#39;s instructions.</li>
		<li>Using tweezers, grip the uppermost edge of an insert containing cell type #1 and gently place it in the appropriate well containing cell type #2.</li>
		<li>After transferring all inserts, check for the presence of air bubbles beneath the membrane of inserts. 
		NOTE: Air bubbles prevent any exchange across the membrane of the insert and can jeopardize the entire experiment.</li>
		<li>If air bubbles are present, very gently lift the insert from the well using tweezers and plunge back into the cell culture medium. The bubbles will disappear. If they are still present, try gently dipping inserts back into the cell culture medium at an angle. 
		NOTE: Do not knock or stir inserts to avoid dissociating adherent cells.</li>
		<li>After removing all the air bubbles and checking that the volumes of media in both compartments, place the lid on the plate and incubate.</li>
	</ol>
	</li>
	<li>Refreshing media in a co-culture system 
	NOTE: Although the membrane readily allows media exchanges between insert and well, refreshing the medium is done in both compartments since the time required to reach equilibrium in the upper and lower compartments by diffusion alone can be quite long.
	<ol>
		<li>Refreshing medium in inserts containing cell type #1 is done in the same way as in step 1.3).</li>
		<li>To refresh medium in wells containing cell type #2, gently slide the insert to the side to create a space wide enough to accommodate a pipette tip. Refresh the medium as in step 1.4).</li>
		<li>Check for the presence of air bubbles and verify the volumes as per steps 1.5.3) through 1.5.5).</li>
	</ol>
	</li>
</ol>
2. Example: measuring the effects of cytokines secreted by LPS-activated N9 microglia on neuronal PC12 cells
NOTE: The following steps are designed for specific flask, well and dish sizes. However, the protocol can be customized for any plasticware dimensions. For media and composition see Materials Table.
<ol>
	<li>Seeding and differentiating PC12 cells in multiwell tissue culture plates
	<ol>
		<li>Warm routine PC12 cell culture medium, PC12 differentiation medium and trypsin-EDTA in a 37&#176;C water bath.</li>
		<li>Use PC12 cells at 60-80% confluence from a 75 cm2 flask.</li>
		<li>With a 15 mm Pasteur pipette, perform vacuum aspiration of the entire cell culture medium in the flask.</li>
		<li>Gently rinse the cell monolayer with 5 ml of sterile phosphate buffered saline and remove the liquid with a Pasteur pipette. Be careful not to dissociate cells at this step.</li>
		<li>Cover the cell monolayer with 3 ml of trypsin-EDTA and incubate for 2-3 min at 37&#176;C.</li>
		<li>Ensure that all the cells are detached under a microscope. If very few cells are floating, incubate for a longer period for a maximum of 5 min.</li>
		<li>Add 10 ml of routine PC12 cell culture medium in order to inactivate the trypsin-EDTA.</li>
		<li>With a 10 ml pipette, gently triturate while making sure that most cells are dissociated from the bottom of the flask. Avoid creating air bubbles in the cell suspension.</li>
		<li>With the same 10 ml pipette, transfer the cell suspension in a 50 ml centrifugation tube.</li>
		<li>Centrifuge for 1 min at 3,200 x g</li>
		<li>Discard the supernatant with a Pasteur pipette while taking care not to disturb the pellet.</li>
		<li>Add 10 ml of routine PC12 cell culture medium with a 10 ml pipette. 
		NOTE: This volume can be adjusted if the pellet is unusually small or big.</li>
		<li>Triturate with the same 10 ml pipette to homogenize the pellet. PC12 cells often clump together so at least 20 pipetting up and downare necessary. 
		NOTE: While vigorous triturating is necessary, avoid creating air bubbles in the cell suspension.</li>
		<li>In a 1.5 ml tube, prepare an appropriate dilution of the cell suspension in trypan blue. Count the cells using a hemocytometer according to previously established protocols74.</li>
		<li>In a separate 50 ml tube, split the cell suspension with PC12 differentiation medium to obtain a diluted cell suspension appropriate for seeding wells from a 24-well plate (30,000 &#162;/cm2, 0.6 ml per well according to the manufacturer&#39;s protocol). 
		NOTE: The 24-well plate must be previously coated with collagen as specified by previously established protocols75.</li>
		<li>Distribute 0.6 ml of the cell suspension per well using a micropipette.</li>
		<li>When all the wells are seeded, rock the plate as in step 1.1.7).</li>
		<li>To allow the proper differentiation of PC12 cells, incubate the 24-well plates for 7-9 days at 37&#176;C in a 5% CO2 humid atmosphere in PC12 differentiation medium15,16 before performing co-culture experiments.</li>
		<li>Perform medium changes every other day by removing half of the liquid and replacing it with an equal volume of fresh PC12 differentiation medium.</li>
	</ol>
	</li>
	<li>Seeding N9 microglia in the inserts and treating with LPS
	<ol>
		<li>One day before performing the co-culture experiments, pre-treat PTFE 0.4 &#181;m-pore inserts with routine N9 cell culture medium to optimize cell adherence, following steps 1.1.1) through 1.1.4)</li>
		<li>Warm routine N9 cell culture medium and trypsin-EDTA in a 37&#176;C water bath.</li>
		<li>Meanwhile, weigh approximately 10 mg of LPS in a 1.5 ml tube for later use. 
		NOTE: Since LPS is a potent pro-inflammatory endotoxin and requires particular safety precautions, glasses, gloves, and a particulate respirator are strongly recommended.</li>
		<li>Use N9 cells at 80-90% confluence from a 75 cm2 flask.</li>
		<li>Follow steps 2.1.3) to 2.1.14). Always use routine N9 cell culture medium instead of routine PC12 cell culture medium. Also note that N9 microglia do not clump together as much as PC12 cells do, so fewer pipetting up and down may be necessary at step 2.1.13).</li>
		<li>In a separate 50 ml tube, split the cell suspension with N9 cell culture medium to obtain a diluted cell suspension appropriate for seeding inserts designed to hold in 24-well plates (60,000 &#162;/cm2, 0.05 ml per insert according to the manufacturer&#39;s protocol, for membrane surface area see manufacturer information). 
		NOTE: These inserts are already coated with collagen by the manufacturer and are optimized for cell adhesion.</li>
		<li>Distribute 0.05 ml of the cell suspension per insert using a micropipette.</li>
		<li>When all inserts are seeded, rock the plate as in step 1.1.7).</li>
		<li>Perform serial dilutions of LPS using N9 treatment medium to obtain 4 &#181;g/ml, 2 &#181;g/ml and 1 &#181;g/ml&#160;working solutions.</li>
		<li>Pipette 0.05 ml of the 4 &#181;g/ml working solution in one the set of inserts in order to yield a final dilution of 2 &#181;g/ml.</li>
		<li>Repeat step 2.2.10) for the other two working solutions in different sets inserts, yielding final dilutions of 1 &#181;g/ml and 0.5 &#181;g/ml respectively.</li>
		<li>Incubate the plates containing the inserts for 24 hr at 37&#176;C in a 5% CO2 humid atmosphere to allow N9 microglia activation with LPS before performing the co-culture experiments.</li>
	</ol>
	</li>
	<li>Co-cultivating neuronal PC12 cells with N9 microglia
	<ol>
		<li>At 7-9 days of differentiating PC12 cells, activate the N9 microglia after 24 hr of incubation with LPS by warm N9 treatment medium and PC12 treatment medium in a 37&#176;C water bath.</li>
		<li>Perform a total medium change for N9 microglia as in step 1.3), replacing the entire used medium by 0.1 ml of N9 treatment medium. Do this for every insert.This is necessary to remove all traces of LPS, leaving only activated N9 microglia in the inserts.</li>
		<li>Perform a total medium change for neuronal PC12 cells as in step 1.4). Transfer the inserts into the 24-well plates as in step 1.5).Incubate the N9-PC12 co-cultures for 24 hr or 48 hr at 37&#176;C in a 5% CO2 humid atmosphere.</li>
		<li>After 24 hr or 48 hr, harvest the supernatant and/or the cells for cytotoxicity, enzyme-linked immunosorbent assay (ELISA), Western blot, or other assays.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The most critical step of any insert co-culture system experiment actually dwells in choosing the proper insert to use. Pore size and membrane material must be taken into thorough account, without forgetting to consider the type of cells that will be seeded and the purpose of the experiment. For example, chemotaxic assays may use the same type of membrane than cell co-cultures to analyze cell behavior modulations induced by secreted soluble factors in the absence of cell-cell contact. However, both types of experiments r...

The study of tissues, organs or systems in vitro is an attempt to simplify the very complex relationships existing between the several cellular subtypes that comprise multicellular organisms. Indeed, in vitro studies make it possible to acquire a detailed understanding of single cell populations. There are two major advantages of conducting in vitro experiments: 1) reduced cellular interactions, and 2) the ability to readily manipulate the cellular environment. Hence, these two advantages have allowed scientists to predict the behavior of specific cell types in vivo, leading to the ability to regulate outcomes of extrinsic influences in whole organisms. In that sense, in vitro cell culture often works as a bridge connecting basic and applied life sciences. Nonetheless, there are also several disadvantages of working in vitro, the most important one being that a certain reservation may dwell in the physiological relevance of observed phenotypes. Indeed, when a single cell type is grown in a vessel, the culture loses, to a various extent, its cell-cell connections with other cell types, its contribution to the humoral environment from the tissue and organism of origin, and the anchors within the tissue that enabled it to uphold a particular three-dimensional structure sometimes crucial for cell function.
The question of cell-cell relationships has been addressed by the development of mixed culture techniques. In this method, two or more cell populations are grown together in the same culture vessel. However, these mixed cultures bear important inconveniences. On one hand, some cell subtypes do not physically interact with one another in the tissue of origin and rely solely on paracrine communications sustained by secreted soluble factors and nearby receptors. This is the case for several inflammatory processes that depend on proximal cytokine signaling. In mixed cultures, physical interactions are unavoidable and make it impossible to study paracrine communications in the absence of cell-cell contacts that can yield altered results. On the other hand, achieving cell-specific interpretations from within a mixed population becomes unfeasible without the use of harsh separation techniques that could significantly affect results.
To solve these important issues, the use of conditioned media has been developed as a technique allowing for compartmentalized cultures and the study of paracrine signaling. This method requires the transfer of the supernatant of one cell type, thus named conditioned medium, to wells containing another population of cells. Yet, an important drawback is that short-lived molecules do not survive long enough in the conditioned medium to be transferred to the wells of the second population of cells. Even long-lived molecules will be greatly diluted over time due to diffusion. Furthermore, both cell populations only participate in unidirectional paracrine communication rather than in active bidirectional communication. This leads to the absence of feedback signaling that is vital in recreating accurate multicellular relationships as they exist in vivo.
As a consequence and driven by the need to better simulate the original in vivo conditions in the in vitro cellular environment, several advances in cell culture techniques have been achieved over the years. One of the most significant advancements has been the use of permeable supports with microporous membranes for compartmentalizing cell cultures, used for the first time by Grobstein in 19531. Such permeable supports have been tailored over the years to accommodate numerous cell types and to be used in several different applications. Nowadays, these supports exist as hollow inserts that are designed to rest in wells from a multiwell tissue culture plate or in circular dishes. In a co-culture system, the insert contains one cell type whereas the well or dish contains the other cellular population, allowing to study the contribution of two different populations of cells on their humoral environment (Figure 1). As a result, cellular polarity (basolateral vs apical secretion or signal reception) is preserved, thus conferring insert co-culture systems an important advantage over mixed cultures and conditioned medium techniques. Several types of membrane materials are available, the most common ones being polyester (PET), polycarbonate (PC) or collagen-coated polytetrafluoroethylene (PTFE), and they exist in different pore sizes ranging from 0.4 &#181;m to 12.0 &#181;m. These varieties of materials and pore sizes offer a spectrum of inserts exerting variable features relevant to optical properties, membrane thickness and cell adherence that make them practical at different levels for the following uses not limited to: 
-studying cell differentiation, embryonic development, tumor metastasis and wound repair by chemotaxic assays through permeable membranes; 
-evaluating drug penetration by assessing their transport through epithelial or endothelial monolayers cultured on permeable supports, and; 
-performing cell co-cultures to analyze cell behavior modulations induced by secreted soluble factors in the absence of cell-cell contact.
The purpose of this article is to describe general methodological guidelines to fulfill the third function stated above, that is to evaluate cellular changes mediated by secreted soluble factors in the absence of cell-cell contact using an insert co-culture system. Several different fields of research make use of insert co-culture systems in order to answer questions relevant to the effect of secreted soluble factors on populations of cells. Indeed, paracrine signaling that modulates cellular behavior at various levels is pertinent in all tissues and systems, which makes insert co-culture systems indispensible to ensure advances in these fields. Conversely, the use of inserts can confirm that signal transduction is by direct cell-cell contact and not by secreted factors. One of the most important uses of inserts is in inflammation studies2-14 where the effect of secreted cytokines is evaluated in various cellular players of immunity. In particular, the study of inflammation in the central nervous system (CNS) has greatly profited from insert co-culture studies, which have allowed to better defining the distinct paracrine roles of neurons and microglia in driving neuroinflammation15-21. These systems were also devised to study the anti-inflammatory potential of molecules that relies on their ability to reduce or inhibit the secretion of pro-inflammatory factors22-26. Research pertaining to cancer27-31, in particular the mechanisms underlying angiogenesis32-34 and inflammation35-42 in tumorigenesis, also benefits from insert co-culture systems. Moreover, soluble factors are of prime importance in the processes that drive differentiation and several studies have used inserts to answer questions in that particular field43-50. In the CNS, seeing as neural tissue has a very limited renewal potential, the study of neurotrophism and neuroprotection is fundamental and has been widely ensured by the use of stem cells in co-culture systems51-56. In addition, inserts are also utilized in as diverse fields as nephrology57,58, endothelial interactions and angiogenesis59-62, apoptosis signaling63-65, inflammation in obesity and metabolic syndrome22,23,66-67, inner ear hair cell protection68,69, and even in fungus virulence70,71 and parasitology72,73.
This article offers general methodological guidelines in order to set up an experiment in view of evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. In particular, we will focus our attention on nerve cell co-cultures and their uses in studying neuroinflammatory process. Given the very vast spectrum of experiments that inserts make possible to pilot, it is unbearable to cover every aspect of this cell culture technique. As an example, a specific protocol to measure the effects of cytokines secreted by lipopolysaccharide (LPS)-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

<table border="0" cellpadding="0" cellspacing="0" width="750">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RPMI-1640 medium</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">R8755</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Nutrient Mixture F-12 Ham</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D6421</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use, must be supplemented with 0.365 gm/L L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Horse serum</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">30-2040</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Fetal bovine serum</td>
			<td style="width:71px;">MultiCell</td>
			<td style="width:119px;">80350</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:232px;">Nerve Growth Factor-7S from murine submaxillary gland</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">N0513</td>
			<td style="width:328px;">Reconstitute the lyophilized powder in a solution of buffered saline or tissue culture medium containing 0.1&#8211;1.0% bovine serum albumin or 1-10% serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T3924</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Lipopolysaccharides from&#160;Escherichia coli&#160;055:B5</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">L2880</td>
			<td style="width:328px;">Toxic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Cell culture inserts for use with 24-well plates</td>
			<td style="width:71px;">BD Falcon</td>
			<td style="width:119px;">353095</td>
			<td style="width:328px;">0.4 &#956;m pores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">24-well plates</td>
			<td style="width:71px;">TrueLine</td>
			<td style="width:119px;">TR5002</td>
			<td style="width:328px;">Coat with collagen before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Routine PC12 cell culture medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Routine N9 cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160; 85% RPMI medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 90% DMEM-F12 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160; 10% heat-inactivated horse serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 10% heat-inactivated horse serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160; 5% heat-inactivated fetal bovine serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC12 differentiation medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>N9 treatment medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 99% RPMI medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 99% DMEM-F12 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1% heat-inactivated fetal bovine serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 1% heat-inactivated horse serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 50 ng/mL nerve growth factor</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC12 treatment medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 99% RPMI medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1% heat-inactivated fetal bovine serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an insert co-culture system of two cellular types in the absence of cell-cell contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

The use of insert co-culture systems is particularly pertinent in the study of neuroinflammatory processes that showcase paracrine relationships between different cellular players of the CNS. Immunity in the CNS is accomplished mainly by resident cells called microglia that monitor their environment in their resting ramified state (Figure 2A) and are capable of sensing disturbances that could trouble the very precious homeostasis necessary for proper neuronal function<sup...

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Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

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27.7K Views.  University of Québec. In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Video: Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

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A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Two Types of Assays for Detecting Frog Sperm Chemoattraction

A System for ex vivo Culturing of Embryonic Pancreas

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

An Ex vivo Culture System to Study Thyroid Development

Establishment of an Extracellular Acidic pH Culture System