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1. Culture media and tool preparation
<ol>
	<li>Spray the bench and work area with 70% ethanol and let dry.</li>
	<li>Spray the dissection tools, forceps, and two glass watch dishes, with 70% ethanol and let them dry on the bench.</li>
	<li>Make the supplemented Schneider&#39;s media (SSM, Table 1) and place it on ice.</li>
	<li>Pipette 1 mL of SSM into each of the glass watch dishes.</li>
	<li>Using a micropipette with a sterile tip, transfer the freshly hatched larvae from the plate of PBS to the SSM in the first glass watch dish. Using a micropipette with a sterile tip, transfer the freshly hatched larvae to the SSM in the second glass watch dish.</li>
</ol>
2. Dissections and brain cultures
<ol>
	<li>Once the larvae are in the second glass watch dish with SSM, dissect the brains out of the larvae using forceps and a dissecting microscope. Adjust the magnification as needed.</li>
	<li>Use one forceps to grab the mouth hooks and with the other, gently grab the body halfway down and pull in the opposite direction (Figure 1) to split the larva into two pieces. 
	NOTE: The brain will be located right behind the mouth hooks. Note that there may be other tissues surrounding the brain. Be very careful when removing these tissues as it can result in damaging the brain.</li>
	<li>Once 15-20 brains have been dissected, add 1 mL of SSM into one well of a sterile 12-well culture tray. Transfer the freshly dissected brains into the SSM using a micropipette and a sterile tip (Figure 2A).</li>
	<li>Place the brains in the SSM media in the 12-well culture tray into an incubator at 25 &#176;C for 24 h (Figure 2A).</li>
</ol>
Table 1: Recipe for making supplemented Schneider&#39;s media (SSM). The table lists the volume of all ingredients required for preparing 5 mL of SSM.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ingredient (starting concentration)</td>
			<td>To make 5 mL</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Drosophila media</td>
			<td>3.89 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine Serum (100%)</td>
			<td>500 &#181;L&#60;</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Glutamine (200 mM)</td>
			<td>500 &#181;L</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>Penicillin (5000 units/mL), Streptomycin (50,000 &#181;g/mL)</td>
			<td>100 &#181;L</td>
			<td>1000 U/mL Pen, 1 mg/mL Strep</td>
		</tr>
		<tr>
			<td>Insulin (10 mg/mL)</td>
			<td>10 &#181;L</td>
			<td>0.02 mg/mL</td>
		</tr>
		<tr>
			<td>glutathione (50 mg/mL)</td>
			<td>5 &#181;L</td>
			<td>0.05 mg/mL</td>
		</tr>
		<tr>
			<td colspan="3">In a sterile hood, using sterile technique, add all ingredients together in a 14 mL conical tube. Place it on ice until use.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#181;L Pipette tips</td>
			<td>Denville Sci</td>
			<td>P2102</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L Pipette tips</td>
			<td>Denville Sci</td>
			<td>P2103-N</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L Pipettor</td>
			<td>Gilson</td>
			<td>P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well multiwell culture plates</td>
			<td>Fisher Scientific</td>
			<td>50-197-4477</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-100A</td>
			<td>Grape Plate Ingredients</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof (100%), Decon Labs, 1 gallon bottle</td>
			<td>Fisher Scientific</td>
			<td>2701</td>
			<td>Used to wash off the larvae before the 24 hr hold in culture medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (10%)</td>
			<td>Sigma</td>
			<td>F4135-100ML</td>
			<td>Supplement for cell culture media.</td>
		</tr>
		<tr>
			<td>Fine forceps for dissection</td>
			<td>Fine Science Tools</td>
			<td>11295-20</td>
			<td>Forcepts used in disections. They work best when sharpened.</td>
		</tr>
		<tr>
			<td>Glass Dissection Dish (3 well)</td>
			<td>These are no longer available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>Sigma</td>
			<td>G6013</td>
			<td>Provides oxidative protection during cell culture.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Ensures that the temperature, humidity, and light exposure is exactly the same throughout experiment.</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I0516</td>
			<td>Independant variable of the experiment</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td></td>
			<td></td>
			<td>For aliquoting culture media</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td>Provides support during cell culture</td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Sigma</td>
			<td>P4458-100ml</td>
			<td>Antibiodics used to prevent bacterial contamination of cells during culture.</td>
		</tr>
		<tr>
			<td>Phosphate Buffer, pH7.4</td>
			<td>Made in house</td>
			<td>Made in house</td>
			<td>Solvent used to wash the brains after fixing and staining steps</td>
		</tr>
		<tr>
			<td>Pick</td>
			<td>Fine Science Tools</td>
			<td>10140-01</td>
			<td>Used to pick larvae off of the grape plate</td>
		</tr>
		<tr>
			<td>Schneiders Culture Medium</td>
			<td>Life Tech</td>
			<td>21720024</td>
			<td>Contains nutrients that help the cells grow and proliferate</td>
		</tr>
		<tr>
			<td>Sterile Water</td>
			<td>Autoclave Milli-Q water made in house</td>
			<td></td>
			<td>Needed for Solutions</td>
		</tr>
	</tbody>
</table>

analyzing neural stem cell reactivation in cultured drosophila brain explants

Source: Naomi Keliinui, C., et al. <a href="https://app.jove.com/v/63189">Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants.</a> J. Vis. Exp. (2022).
This video demonstrates a protocol to identify exogenous factors that initiate the reactivation of Drosophila brain neural stem cells from their quiescent state. Freshly hatched larvae are dissected to isolate their brains, which are then cultured in media with or without a test hormone to evaluate the hormone&#39;s potential to induce neural stem cell reactivation and proliferation.

<img alt="Figure 1" src="/files/ftp_upload/22591/22591_Figure1.jpg" /> 
Figure 1: Drosophila larvae in a glass watch dish with SSM. Forceps are properly positioned for dissection. The location of the larval brain (dark grey) is posterior to the mouth hooks (black), and both are shown inside the larva.
<img alt="Figure 2" src="/files/ftp_upload/22591/22591_Figure2.jpg" /> 
Figure 2: </s...

Analyzing Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

1. Culture media and tool preparation

	Spray the bench and work area with 70% ethanol and let dry.
	Spray the dissection tools, forceps, and two glass ...

In response to external stimuli, Drosophila brain neural stem cells, or NSCs, exit quiescence, a state of cell cycle arrest, and reactivate to begin proliferation.
Take freshly hatched Drosophila larvae, a stage where NSCs naturally exit quiescence in vivo.
Grasp the mouth hooks and the body to split the larva.
Locate the brain behind the mouth hooks and excise it.
Transfer the brains to culture plate wells containing media.
In treated wells, the media contains a test hormone, while control wells lack the hormone. Incubate.
Under control conditions, the NSCs remain quiescent due to the absence of external stimuli.
In treated wells, the hormone binds to specific NSC receptors, activating downstream signaling pathways and reactivating the NSCs.
The reactivated NSCs enter the cell cycle and undergo proliferation.
Post-incubation, the brains are now ready for downstream analysis to visualize NSC proliferation, confirming the hormone&#39;s reactivation potential.

analyzing-neural-stem-cell-reactivation-cultured-drosophila-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

22 visualizações. Fonte: Naomi Keliinui, C., et al. Reativação de células-tronco neurais em explantes cerebrais de Drosophila cultivados. J. Vis. Exp. (2022). Este vídeo demonstra um protocolo para identificar fatores exógenos que iniciam a reativação das células-tronco neurais cerebrais da Drosophila de seu estado quiescente. As larvas recém-eclodidas são dissecadas para isolar seus cérebros, que são então cultivados em meio com ou sem um hormônio de teste para avaliar o potencial do hormônio par...

Vídeo: Analisando a reativação de células-tronco neurais em explantes cerebrais de Drosophila cultivados - Conceito

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Indução direta de Neurais Human células-tronco de sangue periférico células progenitoras hematopoéticas

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

JoVE Journal

Biologia do Desenvolvimento

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Um ensaio de Neurosphere para avaliar a ativação de células-tronco neurais endógena em um modelo do rato da lesão medular mínima

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Neurociência

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) has been utilized to investigate replicative/S-phase progression, whereas antibodies against phospho-histone H3 have been utilized to mark mitotic nuclei and cells. A combination of both labels would enable the classification of G0/G1 (Gap phase), S (replicative), and M (mitotic) phases and serve as an important tool to evaluate the effects of mitotic gene knockdowns or null mutants on cell cycle progression. However, the reagents used to mark EdU-labelled cells are incompatible with several secondary antibody-fluorescent tags. This complicates immunostaining, where primary and tagged secondary antibodies are used to mark pH3-positive mitotic cells. This paper describes a step-by-step protocol for the dual-labeling of EdU and pH3 in Drosophila larval neural stem cells, a system utilized extensively to study mitotic factors. Additionally, a protocol is provided for image analysis and quantification to allocate labeled cells in 3 distinct categories, G0/G1, S, S&gt;G2/M (progression from S to G2/M), and M phases.

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

Cell cycle analysis with 5-ethynyl-2'-deoxyuridine (EdU) and phospho-histone H3 (pH3) labeling is a multi-step procedure that may require extensive optimization. Here, we present a detailed protocol that describes all steps for this procedure including image analysis and quantification to distinguish cells in different cell cycle phases.

5-Ethynyl-2'-Desoxyuridine/Phospho-Histone H3 Protocolo de rotulagem dupla para análise de progressão do ciclo celular em células-tronco neurais Drosophila

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) ...

Analyzing Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

Transcrição

Analyzing Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants