Technical Reference #1873

1873.

Mammalian Par3 Regulates Progenitor Cell Asymmetric Division via Notch Signaling in the Developing Neocortex Ronald S. Bultje; David R. Castaneda-Castellanos; Lily Yeh Jan; Yuh-Nung Jan; Arnold R. Kriegstein; and Song-Hai Shi, Memorial Sloan Kettering Cancer Center, Neuron, 63(1873), (2009)
Link To Paper

Materials & Methods:
Organotypic Cortical Slice Culture and Time-Lapse Confocal Imaging- Twelve hours after in utero electroporation embryos were removed and the brains were extracted into ice-cold artificial cerebro-spinal fluid (ACSF) containing (in mM): 125 NaCl 5 KCl 1.25 NaH2PO4 1 MgSO4 2 CaCl2 25 NaHCO3 and 20 glucose; pH 7.4 310 mOsm/l. Brains were embedded in 4% low-melting agarose in ACSF and sectioned at 400 mm using a vibratome (Leica microsystems). Brain slices that contained EGFP-expressing cells were then transferred onto a slice culture insert (Millicell) in a glass-bottom petri dish (MatTek Corporation) with culture medium containing (by volume): 66% BME 25% Hanks 5% FBS 1% N-2 1% Penicillin/Streptomycin/Glutamine (all from GIBCO) and 0.66% D-(+)-glucose (Sigma). Cultures were maintained in a humidified incubator at 37 C with constant 5% CO2 supply. Twelve hours after plating petri dishes with slice cultures were transferred to an inverted confocal microscope (LSM 5 Pascal Carl Zeiss) and time-lapse images of EGFP-expressing cells were acquired every 3–4 hr for about 24 hr. Transmitted- light images were also taken at each time point to monitor the movement of EGFP-expressing cells in relation to the ventricular surface. Slices were kept in the incubator between time points. After imaging slices were rinsed once in PBS and fixed in 4% PFA for 24 hr and processed for immunohistochemistry using an anti-Tbr2 antibody (Millipore/Chemicon). EGFP-expressing cells that divided unambiguously during the imaging period were identified for the analysis of cell division location and mode.

Microscopic Technique
Inverted Confocal Microscopy

Cell Type(s)
Brain slices that contained EGFP-expressing cells