Name: an enhanced green fluorescence protein-based assay for studying neurite outgrowth in primary neurons
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Description: Watch this Scientific Journal Video about An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons at JoVE.com

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 rins...

<ol>
	<li>Kaplan, A., Bueno, M., Hua, L., Fournier, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+functional+axon+repair+in+the+injured+central+nervous+system:+Lessons+from+neuronal+development.">Maximizing functional axon repair in the injured central nervous system: Lessons from neuronal development.</a> Developmental Dynamics. 247 (1), 18-23 (2018).</li><li>Harrill, J. A., Mundy, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+neurite+outgrowth+in+PC12+cells.">Quantitative assessment of neurite outgrowth in PC12 cells.</a> Methods in Molecular Biology. 758, 331-348 (2011).</li><li>Yeyeodu, S. T., Witherspoon, S. M., Gilyazova, N., Ibeanu, G. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+functional+expression+of+NMDA+receptors+in+PC12+cells.">Lack of functional expression of NMDA receptors in PC12 cells.</a> Neurotoxicology. 28 (4), 876-885 (2007).</li><li>LePage, K. T., Dickey, R. W., Gerwick, W. H., Jester, E. L., Murray, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+neuro-2a+neuroblastoma+cells+versus+intact+neurons+in+primary+culture+for+neurotoxicity+studies.">On the use of neuro-2a neuroblastoma cells versus intact neurons in primary culture for neurotoxicity studies.</a> Critical Reviews in Neurobiology. 17 (1), 27-50 (2005).</li><li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> Journal of Neuroscience. 30 (18), 6171-6177 (2010).</li><li>Cheung, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FE65+interacts+with+ADP-ribosylation+factor+6+to+promote+neurite+outgrowth.">FE65 interacts with ADP-ribosylation factor 6 to promote neurite outgrowth.</a> The FASEB Journal. 28 (1), 337-349 (2014).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+adaptor+FE65+stimulates+Rac1-mediated+neurite+outgrowth+by+recruiting+and+activating+ELMO1.">Neuronal adaptor FE65 stimulates Rac1-mediated neurite outgrowth by recruiting and activating ELMO1.</a> Journal of Biological Chemistry. 293 (20), 7674-7688 (2018).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. 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D., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochalasin+D+inhibits+actin+polymerization+and+induces+depolymerization+of+actin+filaments+formed+during+platelet+shape+change.">Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change.</a> Nature. 293 (5830), 302-305 (1981).</li><li>Calabrese, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+and+regulating+neurite+outgrowth.">Enhancing and regulating neurite outgrowth.</a> Critical Reviews in Toxicology. 38 (4), 391-418 (2008).</li><li>Lau, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexras1+Interacts+with+FE65+to+Regulate+FE65-Amyloid+Precursor+Protein-dependent+Transcription.">Dexras1 Interacts with FE65 to Regulate FE65-Amyloid Precursor Protein-dependent Transcription.</a> Journal of Biological Chemistry. 283 (50), 34728-34737 (2008).</li><li>Cui, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niacin+treatment+of+stroke+increases+synaptic+plasticity+and+axon+growth+in+rats.">Niacin treatment of stroke increases synaptic plasticity and axon growth in rats.</a> Stroke. 41 (9), 2044-2049 (2010).</li><li>Khodosevich, K., Monyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+involved+in+neurite+outgrowth+of+postnatally+born+subventricular+zone+neurons+in+vitro.">Signaling involved in neurite outgrowth of postnatally born subventricular zone neurons in vitro.</a> BMC Neuroscience. 11, 18 (2010).</li><li>Tang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resveratrol+Enhances+Neurite+Outgrowth+and+Synaptogenesis+Via+Sonic+Hedgehog+Signaling+Following+Oxygen-Glucose+Deprivation/Reoxygenation+Injury.">Resveratrol Enhances Neurite Outgrowth and Synaptogenesis Via Sonic Hedgehog Signaling Following Oxygen-Glucose Deprivation/Reoxygenation Injury.</a> Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology. 43 (2), 852-869 (2017).</li><li>He, W., Liu, Y., Tian, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosuvastatin+Improves+Neurite+Outgrowth+of+Cortical+Neurons+against+Oxygen-Glucose+Deprivation+via+Notch1-mediated+Mitochondrial+Biogenesis+and+Functional+Improvement.">Rosuvastatin Improves Neurite Outgrowth of Cortical Neurons against Oxygen-Glucose Deprivation via Notch1-mediated Mitochondrial Biogenesis and Functional Improvement.</a> Frontiers in Cellular Neuroscience. 12, 6 (2018).</li><li>Tesarova, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+for+advanced+glycation+end+products+(RAGE)--soluble+form+(sRAGE)+and+gene+polymorphisms+in+patients+with+breast+cancer.">Receptor for advanced glycation end products (RAGE)--soluble form (sRAGE) and gene polymorphisms in patients with breast cancer.</a> Cancer Investigation. 25 (8), 720-725 (2007).</li><li>Park, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Research. 126 (3), 397-425 (1977).</li><li>Banker, G. A., Cowan, W. 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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 th...

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 1<stro...

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 ...

Low transfection efficiency is a major obstacle to determining how protein reactivates neurite outgrowth in primary neurons. Our protocol overcome this issue by co-transfection of EGFP to identify successfully transfected cells. The high co-transfection rate of EGFP and the proteins of interest ensures the accuracy of the assay.And consequently, immunofluorescence staining is now required. As neurite outgrowth occurs during neural regeneration, this method can be applied to the identification of novel targets for restoring the damaged neuronal network in brain trauma or neurodegenerative diseases. Before beginning the procedure, place a sterile 18 millimeter circular coverslip into each well of a 12 well tissue culture plate and coat each coverslip with five micrograms per millimeter of Poly-D-Lysine solution in a humidified 37 degree Celsius incubator for at least one hour.At the end of the incubation, aspirate the Poly-D-Lysine solution and rinse the coverslips with sterile water. On embryonic day 18, place the uterus harvested from a timed pregnant Sprague Dawley rat into a 10 centimeter Petri dish and use small dissecting scissors to open the uterus and the amniotic sac. Use the scissors to remove the placenta and use forceps to transfer one whole embryo into a new 10 centimeter Petri dish containing chilled PBS glucose.Use the scissors to cut along the sagittal suture of the skull and use a small flat spatula to transfer the embryonic brain into a new 10 centimeter Petri dish containing fresh ice cold PBS glucose. Using two number five tweezers and a dissection microscope, separate the two cerebral hemispheres from the cerebellum and brain stem and remove the meninges. Then isolate the cortex from the cerebral hemispheres and place the cortex in a 15 milliliter tube containing fresh ice cold PBS glucose.For primary cortical neuron culture, in a class II biosafety cabinet, allow the cortex to settle by gravity for five minutes at four degrees Celsius before replacing the PBS glucose with 0.05%trypsin EDTA. Mix with gentle tapping and incubate the tissue in a 37 degree Celsius water bath for 10 minutes with additional gentle tapping every two minutes. At the end of the incubation, stop the digestion with four milliliters of maintenance medium and use a one milliliter pipette to dissociate the tissue with gentle trituration.Sediment the cells by centrifugation and resuspend the pellet in one milliliter of maintenance medium for a second centrifugation. Resuspend the pellet formed after the second centrifugation in one milliliter of fresh maintenance medium for counting and plate the isolated neurons at a 6.5 times 10 to the fourth viable cells per centimeter squared concentration in one milliliter of maintenance medium per well in a 12 well plate. After two days at 37 degrees Celsius and 5%carbon dioxide, dilute EGFP and target protein plasmid DNAs and transfection reagent in two separate 1.5 milliliter tubes and then mix them together.Transfect the cells with EGFP construct by adding the transfection mix to the wells. After 24 hours, wash the cells with 37 degrees Celsius PBS and fix them with 4%paraformaldehyde in PBS for 10 minutes in the dark at room temperature. At the end of the incubation, wash the fixed cells three times with fresh PBS per wash and add a minimal volume of fluorescence mounting medium onto one microscope glass slide per sample.Then carefully transfer the coated coverslips onto the mounting media with the samples facing the glass slides. To image the neurite growth, select the 40X objective on an epifluorescent microscope and click start to capture images from at least 40 intact EGFP positive neurons per transfection. When all of the neurites have been imaged, open the captured images in ImageJ with the NeuronJ plugin and measure the length of the longest neurite of each neuron from the cell body to the tip of the growth cone.Then analyze the data obtained with the software to determine the effect of the targeted proteins on neurite growth. Cytochalasin D suppresses neurite extension in a dose-dependent manner while neurite outgrowth is enhanced in neurons treated with nerve growth factor. In addition, the neuronal growth adaptor protein FE65 significantly stimulates neurite outgrowth two-fold compared to untransfected neurons.Despite a low transfection efficiency, immunofluorescence analysis reveals that over 80%of a two-day cell culture can be successfully co-transfected with EGFP and FE65. EGFP expression is detected at six hours post transfection while FE65 is first detected at hour 12 and both signals are detected for up to seven days post transfection. The transfection of primary rat cortical neurons with different amounts of FE65 plasmid DNA reveals a dose-dependent increase in neurite extension with 0 to 0.5 micrograms of FE65 plasmid.However, no significant difference in growth is observed in neurons transfected with 0.5 or one micrograms of the plasmid. When attempting the protocol, it is important to isolate the appropriate region from the embryonic brain. This technique can also be applied to the study of neural growth in human iPSC-derived neurons which have valuable tools for regenerative medicine.

Research

JoVE Journal

Developmental Biology

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

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 ...

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

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 ...

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 ...

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

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 ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

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 ...

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

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 ...

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 ...

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 ...

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 ...

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

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 ...