NeuroCult™ Basal Medium (Mouse & Rat)

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Publication protocol

Neurosphere formation assays. The procedures were carried out in the tissue-culture hood unless indicated otherwise. All the surgical instruments and solutions used were sterilized. Briefly, the pregnant mice at E16.5 or E18.5 were euthanized and embryos were dissected out and put in PBS. Heads was collected, washed in PBS and kept in PBS/2% glucose on ice for subsequent manipulation. Under a bench dissecting microscope, the head skin was removed and the skull was opened with a pair of surgical scissors. The brain was dissected out using a fine pair of forceps in cold PBS/2% glucose solution. The cerebral cortices were segregated from the rest of the brain with the forceps under the dissecting microscope and collected into an Eppendorf tube containing 1 ml of the neurosphere complete culturing medium (NeuroCult NSC Basal Medium, StemCell Technologies, #05700) supplemented with 10% NeuroCult Proliferation Supplement (StemCell
Technologies, #05701) and 20 ng/ml rhEGF (StemCell Technologies, #78006.1) on ice. The tube was transferred into the hood for pipetting up and down gently for 8 times with a 1 ml Pipetman tip to prepare single cell suspension, with the Pipetman set at 0.8 ml. Air bubbles was minimized during pipetting as excessive oxygen from the bubbles was deleterious for subsequent NSC growth. After pipetting, the tube was kept still at room temperature for 1 min and the supernatant was transferred to a new tube for centrifugation in a bench-top centrifuge for 5 min at 150 g. The supernatant was discarded, and the pellet was gently mixed with 1 ml of the fresh neurosphere complete culture medium. The suspension was filtered through a sterile 40-μm nylon cell strainer (Fisher Scientific, #22363547) and the resulting filtrate was considered as the single-cell preparation. After counting, wild-type and mutant cells were seeded into wells of a 6-well plate at the same number.
For treatment with the HDAC3 inhibitor RGFP966 (SelleckChem, S7229), different concentrations were used (0, 0.08 or 0.8 μM). At different timepoints, the size, number and cell amount of neurospheres were measured. For size measurement, images were randomly taken under a macroscope. For counting neurospheres, a small aliquot of cultured neurospheres was diluted in a 96-well plate and the neurosphere number was counted under a regular light microscope. For determination of the total cell number, an aliquot of neurospheres was taken and pipetted up and down to prepare single cell suspension for counting. For RNA-Seq, fresh neurospheres were washed once with PBS for subsequent RNA preparation. For immunoblotting, neurospheres were washed once with PBS for extract preparation in the RIPA buffer as described above.
For immunostaining, cultured neurospheres were collected into a 15 ml tube and kept for 10 min in a hood. The supernatant was aspirated and 4% PFA was added for incubation at room temperature for 10 min. After fixation, the upper supernatant was removed and neurospheres were washed trice in PBS (to preserve neurosphere morphology, all separation steps were carried out by standing still in the hood for 10 min, instead of centrifugation). Cells in neurospheres were permeabilized in 0.2% Triton-X for 10 min and blocked in 5% BSA for 30 min, followed by incubation with a primary antibody for 30-60 min, extensive washing and subsequent incubation in the blocking buffer with the respective fluorophore-conjugated secondary antibody for 60 min. Neurospheres were then transferred to a 6- or 12-well plate for examination under a fluorescence microscope.


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