DTT in nuclei isolation

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Hi,

I am working with isolation of nuclei from human prefrontal cortex.

The protocol I am currently using can be summarized as follows: tissue homogenization using dounce homogenizer followed by a clean-up that is carried out with gradient centrifugation (nuclei suspension is layered on top of a gradient, which is on top of a cushion. After centrifugation the nuclei are collected at the gradient-cushion interphase).
Homogenization buffer, gradient and cushion all contain beta-mercaptoethanol at a concentration of 1 mM.
I would like to substitute beta mercaptoethanol with DTT (Dithiothreitol). I was wondering if anybody using DTT in nuclei isolation could suggest which concentration I should use.

Thank you

Martina

2

Hi Martina,

We use 0.1mM DTT in our homogenization buffer for nuclei isolation. We do not routinely use gradient centrifugation for prep cleanup. You can find our SOPs for nuclei isolation from human brain tissues on protocols.io at the links below:
Isolation of Nuclei from Adult_Human_Brain_Tissue
Isolation of Nuclei from Adult Human Brain Tissue for 10x Genomics Platform

Best,

Rebecca