Antibiotics antimycotic

Embryonic brains were dissected from 14.5 days post coitum (dpc) and placed in Ca²⁺ and Mg²⁺-free Hank's Balanced Salt Solution (HBSS) to remove the cerebellum and meninges. Isolated brains were transferred to trypsinization solution containing 0.05% trypsin/EDTA (Cellgro) and incubated for 30 min at 37°C with vigorous shaking at 250 rpm. Trypsinization was quenched by adding an equal volume of cell culture medium (DMEM supplemented with 10% fetal bovine serum (FBS), 20 mM L-glutamine, and 1% antibiotics/antimycotics (Cellgro)). After centrifugation at 1,000 rpm for 5 min, the tissue was triturated in neuronal cell culture medium (Neurobasal® medium supplemented with B-27® supplement (Invitrogen), 1× GlutaMax, 0.5 mM L-glutamine, and 1% antibiotics/antimycotics (Cellgro)) by gentle pipetting through a 1000 μl pipette tip. Triturated tissues were strained through a 40 μm nylon mesh. Resulting cells were plated on a tissue culture dish coated with poly-D-lysine (MW 30,000–70,000, Sigma-Aldrich) and laminin (Invitrogen) at 1 × 10⁴ to 1.5 × 10⁵ cells/cm² depending on the experiment, and then cultured in the same medium. One-half of the medium was changed every three days.

Primary astrocytes from the cerebral hemispheres of newborn SD rats were cultured as described previously (Kaech & Banker, 2006). In brief, cortices were triturated into single cells in DMEM (Cat No. SH30243.01; Hyclone, Logan, UT, USA) containing 10% horse serum (Cat No. 16050130; Invitrogen, Carlsbad, CA, USA) and 1% antibiotics–antimycotics (Cat No. 30‐004‐CI; Corning, Inc., Corning, NY, USA) and plated on 75‐cm² poly‐d‐lysine‐coated culture flasks (0.5 hemisphere/flask; BD Biosciences, Franklin Lakes, NJ, USA). After 2 days, unattached cells and debris were removed by changing the medium. Cultures were fed every 2–3 days with fresh medium until they reached 70–80% confluence.
To determine whether BDNF‐treated astrocyte‐conditioned medium (CM) contributes to hippocampal neuronal survival after exposure to thrombin, secondary astrocyte cultures were washed and treated with 80 ng·mL⁻¹ BDNF for 24 hr. The cells were washed three times with PBS and incubated in serum‐free medium for 24 hr. The CM was then collected and centrifuged at 2,000× g for 10 min at 4°C and filtered through a 0.2‐μm filter to remove cell debris. The untreated astrocyte‐CM served as the control. All media were stored at −70°C until use.