This work was supported by funds from the Research Grants Council Hong Kong, Health and Medical Research Fund (Hong Kong), CUHK direct grant scheme, the United College endowment fund and the TUYF Charitable Trust.

All procedures followed were in accordance with the ethical standards of the animal experimentation ethics committee of the Chinese University of Hong Kong.
1. Preparation of Coverslips
<ol>
	<li>Place a sterile 18 mm circular coverslip into each well of a 12-well tissue culture plate.</li>
	<li>Coat the coverslip with 5 &#181;g/mL poly-D-lysine solution in a humidified 37 &#176;C incubator for at least 1 h.</li>
	<li>Aspirate the poly-D-lysine solution from the tissue culture plate and rinse the coated coverslips once with sterile water.</li>
</ol>
2. Rat Embryonic Neuron Dissection
<ol>
	<li>Sacrifice a timed-pregnant Sprague-Dawley rat at a gestational age of 18 days (E18) by either cervical dislocation or CO2 asphyxiation. 
	NOTE: Please check local regulations for the sacrifice of pregnant rats.</li>
	<li>Open the abdominal cavity of the pregnant rat with dissecting scissors and transfer the uterus to a 10 cm Petri dish.</li>
	<li>Open the uterus and the amniotic sac carefully with small dissecting scissors and remove the placenta from the rat embryo using small dissecting scissors. Transfer the whole embryo to a 10 cm Petri dish with pre-chilled phosphate buffered saline supplemented with glucose (PBS-glucose, 10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride and 3 g/L glucose) using a pair of small forceps.</li>
	<li>Cut along the sagittal suture of the skull and open it carefully with a pair of small dissecting scissors. Transfer the embryonic brain with a small flat spatula to a 10 cm Petri dish with ice-cold PBS-glucose.</li>
	<li>Separate the two cerebral hemispheres from the cerebellum and brain stem using two #5 tweezers under a dissection microscope. 
	NOTE: Please see reference12 for the structure of the rat brain.</li>
	<li>Remove the meninges using the #5 tweezers.</li>
	<li>Isolate the cortex from the cerebral hemispheres with two straight #5 tweezers.</li>
	<li>Transfer the isolated cortex to ice-cold PBS-glucose in a 15 mL centrifuge tube.</li>
</ol>
3. Primary Cortical Neuron Culture
NOTE: All procedures in steps 3 and 4 are performed inside a Class II Biosafety cabinet.
<ol>
	<li>Settle the isolated cortex by gravity at 4 &#176;C for 5 min and aspirate the PBS-glucose.</li>
	<li>Add 1 mL of 0.05% Trypsin-EDTA to the isolated cortex and mix gently by tapping and incubate the tissue in a 37 &#176;C water bath for 10 min to allow enzymatic digestion. Tap the tube gently to mixing every 2 min.</li>
	<li>Add 4 mL of maintenance medium (e.g., Neurobasal Medium) to the tissue/trypsin mixture. 
	NOTE: All the maintenance medium used in this protocol is supplemented with Penicillin-Streptomycin and B-27 supplement13.</li>
	<li>Dissociate the tissue gently by trituration using a 1 mL pipette.</li>
	<li>Pellet the dissociated cells by centrifugation at 200 x g for 5 min. Aspirate the supernatant.</li>
	<li>Repeat steps 3.5 to 3.7 twice.</li>
	<li>Resuspend the cell pellet in 1 mL of maintenance medium.</li>
	<li>Add 10 &#181;L of 0.4% Trypan Blue solution to 10 &#181;L of cell suspension for counting of viable cells by a hemocytometer.</li>
	<li>Plate the neurons at a density of 65,000/cm2 (viable cells) in 1 mL of maintenance medium per well in a 12-well plate.</li>
</ol>
4. Cell Transfection and Fixation
<ol>
	<li>At 2 days in vitro (DIV2), transfect 0.5 &#181;g of EGFP construct (pEGFP-C1) to neurons together either with or without of 0.5 &#181;g of POI by using 1 &#181;L of transfection reagent (e.g., Lipofectamine 2000). Use manufacturer&#39;s instructions. 
	NOTE: Mammalian expression constructs were prepared by using an endotoxin free plasmid preparation kit. Treatment with chemicals/molecules (in this manuscript cytochalasin D (Cyto D) and nerve growth factor (NGF) were used) can be done at 6 h after transfection.</li>
	<li>Aspirate the culture medium 24 h post-transfection and wash the transfected cells once with 37 &#176;C PBS (10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride).</li>
	<li>Fix the cells with 4% paraformaldehyde in PBS for 10 min in the dark at room temperature.</li>
	<li>Wash the fixed cells three times with PBS.</li>
	<li>Add a minimal amount of fluorescence mounting medium on a microscope glass slide. Carefully transfer the coverslip from the 12-well plate onto the mounting medium with the sample facing the glass slide. 
	NOTE: Seal the edge of the coverslip with nail polish if an aqueous mounting medium is used.</li>
</ol>
5. Measurement of Neurite Outgrowth
<ol>
	<li>Use a 40x objective for capturing images using an epi-fluorescent microscope.</li>
	<li>Capture images from at least 40 intact neurons with EGFP signal per transfection.</li>
	<li>Open the captured image in ImageJ software with the NeuronJ plugin11 to measure the length of the longest neurite, from the cell body to the tip of the growth cone, of each imaged neuron.</li>
	<li>Analyze the data obtained with the software to determine the effect of the targeted proteins in neurite outgrowth.</li>
</ol>

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The authors declare that they have no conflicts of interest with the contents of this article.

As stated before, PC12 and its subclones are widely employed for studying neurite extension because they have excellent transfection efficiency2,3. In contrast, primary neurons have a low transfection rate, which is a major obstacle for studying neurite outgrowth regulators by transfection7. Here, we describe a convenient protocol for quantifying neurite outgrowth in primary neurons. Despite the low overall transfection efficiency, more than 80% of the transfected neurons were successfully co-transfected with the two proteins: FE65 and EGFP (Figure 2A). Therefore, by analyzing the EGFP labelled neurons, the effect of FE65 on neurite outgrowth could be precisely determined (Figure 2B).
Another advantage of the EGFP-based approach described is that immunofluorescence staining can be omitted. Immunofluorescence staining of Tuj 1, a neuron-specific class III &#946;-tubulin, is widely employed as a morphological marker when studying neurite elongation18,19,20,21. In addition to Tuj 1, staining of the POI is required for the identification of transfected neurons19,22,23. It is known that the consistency of immunofluorescence staining, which is crucial for neurite outgrowth measurement, is affected by many factors including sample preparation and antibodies24. Hence, the overall simplicity of the EGFP-based method could improve the accuracy of the assay.
In our protocol, neurons are fixed for neurite measurement 24 h post-transfection. Thus, neurons are exposed to the transfection reagent for 24 h. It is long known that transfection reagents are toxic to cells25. If the effect of the POI on neurite extension needs to be determined beyond DIV3, fresh medium replenishment may help to minimize the toxicity. Moreover, alternative gene delivery methods may be considered such as Ca2+ phosphate co-precipitation and nucleofection, both of which are shown to have a less toxic effect to cells26. Additionally, we show here that the highest effect of FE65 on neurite outgrowth is observed in the neurons transfected with that 0.5 &#181;g of FE65 and 0.5 &#181;g of EGFP plasmids by using 1 &#181;L of transfection reagent. For other POIs, the optimal amounts of plasmid DNA and transfection reagents should be determined as proteins have widely different turnover rates.
In addition to gene delivery method, cell density is another critical parameter to be considered. If the neuron density is too high, it becomes difficult to identify the origins of neurites as they would overlap with each other. Although plating cells at a low density may resolve the issue, the survival of primary neurons would be significantly reduced at a low cell density27. Primary rat hippocampal neurons have been shown to grow healthily at the density between 40,000 to 100,000/cm2&#160;28. In this protocol, a density of 65,000/cm2 of rat cortical neurons is used. Nevertheless, it is important to determine the appropriate cell density for different types of neurons under different experimental conditions.
The neurite measurement of developing rat primary neurons (i.e.&#160;DIV3 neurons) is described here. However, more mature neuronal phenotypes can be observed in neurons beyond DIV329. As the effects of POIs or drugs on neurite extension in mature neurons may be different from the developing neurons, investigation by using neurons from different stages would provide a more comprehensive perspective. It is worth noting that we were still able to observe EGFP expression in the neurons 7 d post-transfection. This may facilitate the study of POIs or drugs on neurite outgrowth in mature neurons.
Human induced pluripotent stem cell (hiPSC)-derived neurons are valuable tools in identification of novel therapeutic approaches. For instance, investigation of neurite outgrowth in these cells could reveal novel targets in neuroregeneration as the use of hiPSC-derived neurons avoids the species differences. Similar to primary rodent neurons, hiPSC-derived neurons are difficult to transfect30, which may reduce the co-transfection efficiency of EGFP and the POI. Hence, the use of polycistronic mammalian expression vectors could ensure that all the transfected cells express the entire set of transfected genes including EGFP and POIs. Additionally, alternative gene delivery methods, such as electroporation, may improve transfection efficiency. Again, cell viability is an issue which needs to be considered when using such gene delivery approaches.
It is known that the rat embryonic brain cortex contains different types of neurons including spiny stellate neurons, bipolar neurons and long-projecting neurons31. While this protocol stated here can reveal the general effect of POIs on neurite outgrowth, it is possible that the same POI may exhibit different responses in different types of neurons. For example, myostatin increases the number of sensory axons but not that of motor axons32. In such a scenario, modification of the protocol is necessary, such as prior isolation of specific types of neurons by flow cytometry. Alternatively, specific neuronal marker antibodies may be used for the identification of the required types of neurons during imaging.

Neurites, including both axons and dendrites, are the projections from neurons involved in the establishment of the neural networks. The dynamic outgrowth of neurites is essential for neurodevelopment. However, the underlying regulatory mechanisms underneath remain unclear. In particular, neurite damage is often observed in various neurodegenerative diseases and after brain injuries1. Therefore, investigation of the roles of putative molecules in various neurite outgrowth regulatory pathways would improve our understanding of the process. Moreover, it may reveal novel therapeutic targets for various neurological disorders. Neuronal cell lines are valuable models for studying neuronal processes including neurite outgrowth as they are easy to manipulate and transfect2,3. However, genetic drift has been reported to occur in some commonly used cell lines, which could lead to variations in their physiological responses4. Moreover, differential protein expression has been shown between neuronal cell lines and primary neurons. For instance, PC12, a neuronal cell line derived from rat adrenal gland that is widely used for studying neurite outgrowth2,3, does not express NMDA receptors5. Furthermore, it has been proposed that the reduced responsiveness of the mouse neuroblastoma line neuro-2a to neurotoxins in comparison to primary neurons is due to the lack of expression of certain membrane receptors and ion channels6. Therefore, primary neurons are a more desirable and representative model for the investigation of neurite outgrowth. However, the use of primary neurons is hindered by their low transfection efficiency7.
Here, we describe a method that involves the co-transfection of the protein of interest (POI) and EGFP to primary rat cortical neurons. The EGFP acts as a morphological marker for the identification of successfully transfected neurons and permits the measurement of neurites. We validated this method by using compounds/molecules that have been reported to modulate neurite outgrowth. Moreover, FE65, a neuronal adaptor protein that has been shown to stimulate neurite outgrowth, was used to illustrate this approach8,9. This protocol involves (1) the isolation of primary cortical neurons from embryonic day 18 (E18) rat embryos, (2) the co-transfection of neurons with EGFP and the POI (FE65 in this study) and (3) the imaging and analysis of the neurons by using the image processing software ImageJ with the NeuronJ plugin10,11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 tweezers</td>
			<td>Regine</td>
			<td>5-COB</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm Circle Cover Slips</td>
			<td>Thermo Scientific</td>
			<td>CB00180RA</td>
			<td>Sterilize before use.</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin D</td>
			<td>Invitrogen</td>
			<td>PHZ1063</td>
			<td>Dissolved in DMSO.</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors, 10 cm</td>
			<td>World Precision Instruments</td>
			<td>14393</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors, 12.5 cm</td>
			<td>World Precision Instruments</td>
			<td>15922</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Mounting Medium</td>
			<td>Dako</td>
			<td>S302380</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>NGF 2.5S Native Mouse Protein</td>
			<td>Gibco</td>
			<td>13257019</td>
			<td></td>
		</tr>
		<tr>
			<td>Nugent Utility Forceps, 10mm, Straight Tip</td>
			<td>World Precision Instruments</td>
			<td>504489</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>pEGFP-C1</td>
			<td>Clontech</td>
			<td>#6084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>pCI FE65</td>
			<td></td>
			<td></td>
			<td>Please see references 8 and 15</td>
		</tr>
		<tr>
			<td>PBS Tablets</td>
			<td>Gibco</td>
			<td>18912014</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Sigma-Aldrich</td>
			<td>S4147</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

an enhanced green fluorescence protein-based assay for studying neurite outgrowth in primary neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

To test this methodology, we used Cyto D and nerve growth factor NGF, which have been shown to inhibit and stimulate neurite outgrowth respectively14,15,16. The neurite length of neurons transfected with EGFP were measured after treatment with Cyto D or NGF. The transfection efficiency of EGFP to the neurons was 2.7% (1,068 neurons counted). As shown in Figure 1A, Cyto D suppressed neurite extension in a dose-dependent manner. Conversely, neurite outgrowth was potentiated in the neurons treated with NGF (Figure 1B).
Next, we investigated the utility of this system by transfecting the neuronal adaptor FE65, which has been shown to promote neurite outgrowth. Primary rat cortical neurons were co-transfected with FE65 and EGFP. Despite the low transfection efficiency, immuno-fluorescence analysis revealed that over 80% of the neurons were successfully co-transfected with EGFP and FE65 (Figure 2A). Similar to previous reports8,9, FE65 significantly stimulated the neurite outgrowth by 2x (Figure 2B). We also analyzed the expression of EGFP and FE65 at different time points by Western blot analysis. As shown in Figure 2C, EGFP and FE65 were detected 6 h and 12 h post-transfection, respectively. Similar expression levels of the proteins were observed in 1 d to 7 d post-transfection neurons. This indicates that the analysis of neurite outgrowth could be done as early as 6 h post-transfection or in more mature neurons. Together, this data suggest that the mentioned protocol is suitable for determining the role of putative neurite outgrowth regulatory proteins by classical transfection.
We also monitored the effect of gene dosage on neurite outgrowth by transfecting primary rat cortical neurons with different amounts of FE65 plasmid DNA. As shown in Figure 2D, a dose-dependent increase in neurite extension was observed from 0 - 0.5 &#181;g of FE65 plasmid DNA. However, there was no significant difference between neurons transfected with either 0.5 &#181;g or 1 &#181;g of plasmid DNA (Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60031/60031fig1d.jpg" /> 
Figure 1: Neurite outgrowth is modulated by Cyto D and NGF. E18 rat cortical neurons were transfected on DIV2 with an EGFP expression vector. 6 h post-transfection, the cells were treated with (A) 0-0.5 &#181;g/mL Cyto D or (B) 0-100 ng/mL NGF for 24 h. Then the neurons were fixed and imaged accordingly. Images were captured with 40x objective using an epi-fluorescence microscope and the length of the longest neurite from the cell body to the tip of the growth cone was measured by using ImageJ with the NeuronJ plugin. Three independent experiments were performed and at least 40 neurons were measured in each group. The bar chart showed the fold change in mean neurite length. Unpaired t-test was adopted for the statistical analysis. *p &#60; 0.001, **p &#60; 0.05. Error bars = S.E.M. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60031/60031fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60031/60031fig2k.jpg" /> 
Figure 2: FE65 stimulates neurite outgrowth. E18 rat cortical neurons were transfected on DIV2 with either empty vector control (EV) or FE65 together with an EGFP expression vector. Cells were fixed and imaged 24 h after transfection. (A) The transfected neurons were counterstained with anti-FE65 antibody and the number of cells with EGFP or FE65 singly transfected and EGFP + FE65 co-transfected were counted. Three independent experiments were performed and at least 100 cells were counted in each experiment. Data were expressed as the percentage of cells with EGFP and EGFP + FE65 signals. *p &#60; 0.001. Error bars = S.D. (B) The length of the longest neurite from the cell body to the tip of the growth cone was measured by using ImageJ with the NeuronJ plugin. Representative neuron images were shown in the right panel. FE65 was stained by a goat anti-FE65 antibody as described8,17. Three independent experiments were performed and at least 40 neurons were measured per transfection. The bar chart showed the fold change in mean neurite length. The data were analyzed by unpaired t-test. *p &#60; 0.001. Error bars = S.E.M. Scale bar = 10 &#181;m. (C) Western blot analysis of the expression of levels of EGFP and FE65 at the post-transfection time points as indicated. EGFP and FE65 were detected by mouse anti-GFP and anti-myc (to FE65 C-terminal myc tag), respectively. (D) The average neurite length of neurons transfected with various amounts of FE65 plasmid DNA as indicated. Statistical analyses were performed using one-way ANOVA tests with Bonferroni post-hoc test. *p &#60; 0.001, **p &#60; 0.05. Error bars = S.E.M. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60031/60031fig2elarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons at JoVE.com

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.8K Views.  The Chinese University of Hong Kong, Hong Kong Special Administrative Region. In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Video: An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Summary

Explore More Videos

Chapters in this video

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Single-cell Photoconversion in Living Intact Zebrafish

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Induction and Validation of Cellular Senescence in Primary Human Cells

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii