I am trying to dissociate primary neurospheres (mouse), which are cryopreserved in Liq.N2. I followed the standard procedure given by the supplier and the same procedure was followed in many publications. to my wonder i observed that all the neurospheres are getting clumped and Trypan Blue shows all the cells were dead. In continuation, i have diluted the accutase 10 times and i could see improvement in cell viability..my question is why every one else are able to dissociate cells in direct accutase and why i am not able to do it..I wonder why? as per my knowledge no publication mentioned about diluting the accuatse..please clarify my doubt..thanks
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