I am trying to synchronize core-clock gene expression of neuron-enriched cultures from P0 mouse cortex. I am following an accepted method of using 50% serum-containing media, as first described in Balsalobre, 1998, however most literature using this method has focused on fibroblasts.
My problem is:
When performing a 2-hr serum shock followed by 24-40 hours of serum-free media before imaging, the neurons which were present before the serum shock have died and all that is left are glia (astrocytes, based on morphology).
I have since learned that complete media changes can be toxic to cultured neurons, likely accounting for the cell death I'm seeing. Does anyone know of alternative methods for entraining/synchronizing cellular molecular clocks? Temperature entraining may be an option.
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