Name: live imaging of primary cerebral cortex cells using a 2d culture system
Uploaded: 2017-08-09T13:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System at JoVE.com

This work was supported by CNPq (Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico), CAPES (Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior) and FAPERN (Funda&#231;&#227;o de Amparo a Pesquisa do Rio Grande do Norte).

All experiments involving live animals described in this protocol are conducted according to the National and International laws and were approved by the local University Animal Care and Use Committee (CEUA/UFRN), under the license 009/2014. The following protocol is performed in a sterile environment. Familiarity with basic cell culture is expected.
1. Dorsolateral Telencephalon Microdissection
<ol>
	<li>Prepare the dissection medium (100 mL): 98.5 mL Hank&#39;s Balanced Salt Solution (HBSS...

Real time observation of primary cerebral cortex cells allows the analysis of cell proliferation, mode of cell division, cell cycle length, cell differentiation and cell survival3,14,15,37. More importantly, it permits the study of single-cell lineages, leading to the identification of the intermediate phases enacted during the progression from NSCs to neurons3. Finally,...

Neural stem cells (NSC) generate neurons and macroglial cells during cerebral cortex development. At early-corticogenesis, NSCs undergo several rounds of symmetric cell division, and expand the progenitor pool. Then, NSCs divide asymmetrically to generate neurons directly or indirectly through intermediates1. Only at mid- to late-corticogenesis, progenitors switch to generate astrocytes and oligodendrocytes2,3,4. However, the complete mechanisms that control cell proliferation and differentiation, as well as the contribution of fate-restricted progenitors to the generation of unique types of neurons or macroglial cells remain a matter of intense debate4,5,6. The potential of individual cortical NSCs to generate neurons, astrocytes and oligodendrocytes has been extensively studied in vitro and in vivo using a myriad of techniques such as: live imaging in single-cell cultures7,8,9,10,11,12,13; live imaging in high-density cultures cultures3,14,15; live imaging in slice cultures16,17,18; clonal analysis using viral vector-mediated genetic labeling in high-density cultures14,15,19,20,21; clonal analysis in vivo using retrovirus22,23,24,25,26,27,28,29,30,31,32,33; and clonal analysis in vivo using transgenic animals34.
Each of these techniques presents pros and cons. For instance, in vivo lineage tracing is susceptible to the lumping and splitting errors3, leading to conflicting conclusions about the potential of individual cortical progenitors. Moreover, both in vitro and in vivo studies based on the labeling of progenitor cells at early time-points and posterior analysis of cell lineages may be influenced by the undetected occurrence of cell death during lineage-progression35. Therefore, a suitable system to analyze the potential of single NSCs must allow the identification of all cells generated, as well as the appropriate characterization of cell fates within the lineage. Combination of primary cell culture and live imaging provides this setting. Using single-cell culture and time-lapse video microscopy, Temple et al. have shown the switch in the lineage of individual cerebral cortex progenitors from neurogenesis to gliogenesis11. Later, they used the same system to show that different types of neurons are generated from a single cortical progenitor12. However, this system presents an important caveat: only 1% of cortical progenitors isolated at early corticogenesis generate clones of 4 or more progeny9. After the addition of FGF2, the frequency of cells generating 4 or more cells increases to 8 - 10%9. Nevertheless, this number is too small considering that virtually all cortical progenitors are proliferative at this stage. Moreover, the potential effects of FGF2 on fate-specification cannot be ruled out36. To circumvent these limitations, we used high-density cell cultures that support the proliferation of both ventricular (Pax6-expressing) and subventricular (Tbr2-expressing) cortical progenitors15. Moreover, the real-time observation of these cultures has shown that several features of NSC lineage progression are reproduced under these conditions, such as mode of cell division, lengthening of cell cycle, potential of single cells to generate neurons and glia, among others3,15. More recently, we have also used this system to show that CREB-signaling affects the cell survival of immature cerebral cortex neurons in mice37. Thus, we believe that time-lapse video-microscopy of primary murine cerebral cortex cells grown in high density is a powerful and accessible tool to study cellular and molecular mechanisms of cell cycle progression, mode of cell division, cell survival and cell fate specification. The latter can be accomplished using transgenic animals, allowing the identification of specific cell fates on real time38,39,40 or the use of post-imaging immunocytochemistry3,38,41,42.
Here, we provide a step-by-step protocol to prepare primary cerebral cortex cell culture supporting proliferation of NSCs and the subsequent generation of neurons and macroglial cells. We also discuss the use of retroviral-mediated transfection to manipulate gene expression of individual cells, which can be identified and tracked at the single-cell level using time-lapse video microscopy. This protocol can be used to study primary cerebral cortex cells isolated from the beginning to the end of the corticogenesis in rodents, but a few adjustments may be required according to the stage14. NSCs isolated from other sources can also be studied using time-lapse video microscopy of 2D cultures, but the appropriate culturing system should be determined by comparing cell behaviors in vitro and in vivo38,43.

<table width="953">
	<tbody>
		<tr height="40">
			<td height="40" style="width:320px;height:40px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:123px;">Invitrogen Life Technologies</td>
			<td style="width:344px;">14175129</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">H3375-25G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/streptomycin</td>
			<td style="width:123px;">Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:123px;">Gibco</td>
			<td>12400-024</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Calf Serum (FCS)</td>
			<td style="width:123px;">Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glucose</td>
			<td style="width:123px;">Gibco</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B27</td>
			<td style="width:123px;">Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">trypsin-EDTA (0.05%)</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:344px;">25300054</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td style="width:344px;">16005</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:320px;height:40px;">Goat serum</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">69023</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:320px;height:40px;">Triton X-100</td>
			<td style="width:123px;">VWR International Ltd.</td>
			<td style="width:344px;">306324N</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane</td>
			<td>Sigma</td>
			<td>792632</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-MAP2, mouse</td>
			<td>Sigma</td>
			<td>M4403</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-GFP chicken</td>
			<td>Aves</td>
			<td>0511FP12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Goat anti-mouse alexa 594</td>
			<td>Invitrogen</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Goat anti-chicken alexa 488</td>
			<td>Invitrogen</td>
			<td>A11039</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:320px;height:40px;">ImageJ</td>
			<td style="width:123px;">NIH</td>
			<td style="width:344px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">tTt</td>
			<td>ETH Zurich</td>
			<td style="width:344px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell observer microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pasteur pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">The Tracking Tool (tTt) software</td>
			<td></td>
			<td>https://www.bsse.ethz.ch/csd/software/ttt-and-qtfy.html</td>
			<td>download link</td>
		</tr>
	</tbody>
</table>

live imaging of primary cerebral cortex cells using a 2d culture system

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Primary cultures of cerebral cortex cells isolated from embryos E14 contain both progenitor and neuronal cells. During the period of imaging, progenitors undergo several rounds of cell division, increasing the number of cells (Figure 5 and Video Figure 1).
Retroviral-mediated transfection of a few progenitor cells facilitates the identification of cell clones (F...

Watch this Scientific Journal Video about Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System at JoVE.com

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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