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

Summary

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.

Abstract

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.

Introduction

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 oligodendrocytes^2,3,4. However, the complete mechanisms that control cell proliferation and differentiation, as well as the contribution of fate-restricted progenit....

Protocol

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

1. Prepare the dissection medium (100 mL): 98.5 mL Hank's Balanced Salt Solution (HBSS), 0.5 mL 5 M HEPES pH 7.4, 1 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL).
2. Filter-sterilize the dissection medium.

Results

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.......

Discussion

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 survival^3,14,15,37. 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,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hank's Balanced Salt Solution (HBSS)	Invitrogen Life Technologies	14175129
HEPES	Sigma-Aldrich	H3375-25G
Penicillin/streptomycin	Gibco	15140122
Dulbecco Modified Eagle's Medium (DMEM)	Gibco	12400-024
Fetal Calf Serum (FCS)	Gibco	10437028
Glucose	Gibco	A2494001
B27	Gibco	17504044
trypsin-EDTA (0.05%)	Gibco	25300054
Paraformaldehyde	Sigma	16005
Goat serum	Sigma-Aldrich	69023
Triton X-100	VWR International Ltd.	306324N
Isoflurane	Sigma	792632
anti-MAP2, mouse	Sigma	M4403
anti-GFP chicken	Aves	0511FP12
DAPI	Sigma	D9542
Goat anti-mouse alexa 594	Invitrogen	A11005
Goat anti-chicken alexa 488	Invitrogen	A11039
ImageJ	NIH
tTt	ETH Zurich
Cell observer microscope	Zeiss
Pasteur pipette
PBS
The Tracking Tool (tTt) software		https://www.bsse.ethz.ch/csd/software/ttt-and-qtfy.html	download link