The Mechanism of Taurine Protection Against Endoplasmic Reticulum Stress in an Animal Stroke Model of Cerebral Artery Occlusion and Stroke-Related Conditions in Primary Neuronal Cell Culture - Advances in Experimental Medicine and Biology - page 228

23.2 Material and Methods

All animal procedures were carried out in accordance with the Animal Use and
Care Guidelines issued by the National Institutes of Health using a protocol
approved by the Florida Atlantic University, Boca Raton, Animal Use and Care
Committee.

23.2.1 In Vitro Study

23.2.1.1 Primary Neuronal Cell Culture

According to the method of Hartung et al., pregnant rats were sacrificed after
isoflurane exposure, and embryos at 16-18 days were removed. Brains were iso-
lated from the fetuses and placed in Basal Media Eagle (BME) supplemented with
2 mM glutamine, 26.8 mM glucose, and 20% heat-inactivated fetal bovine serum.
This medium is referred to as growth media eagle (GME). The cortices were then
dissociated by passing the tissue through a 14-G cannula. Cells were centrifuged at
300 g/min for 5 min at room temperature. The resulting pellet was resuspended in
GME and plated on appropriate tissue culture plates pre-coated with 5 m g/ml of
poly-D-lysine. Cells were maintained for 1 h in a humidified incubator (37°C, 99%
humidity, and 5% CO 2 ). Incubation medium was replaced with serum-free neu-
robasal medium supplemented glutamine, and the cells were then maintained in an
incubator for 12-18 days until they were ready for handling (Hartung 1998 ) .

23.2.1.2 Hypoxia and Reoxygenation

To generate hypoxic conditions, 14-day-cultured neurons in 6- or 96-well plates
were placed in a hypoxia chamber with oxygen levels maintained at 0.3-0.4%.
The level of oxygen was continuously monitored using an oxygen electrode. Primary
cortical neuronal cultures in the absence or presence of taurine were subjected to
20 h of hypoxia. Reoxygenation was performed by removing cultured plates from
the hypoxic chamber and transferring them into normal culture incubator remaining
for another 20 h.

23.2.1.3 ATP Assay

Primary cortical neuronal cells in 96-well plates were treated with or without tau-
rine (1, 5, and 10 mM) for 1 h, and then cells were subjected to hypoxia-reoxygenation
conditions for 20 h to induce cell death. ATP solution (Promega) was added to each