Counting Isolated Nuclei from Mouse Brain

Written by DeNovix
Cell and tissue biology New products PCR and sequencing

Isolating nuclei is a critical step for single nuclear RNA-sequencing and ATAC-sequencing workflows. Ensuring that non-clustered, debris-free samples are obtained is crucial to successful library preparation. However, nuclei isolated from tissue samples such as a brain have high debris compared to that from cultured cells.

Even when debris does not affect library preparation itself, it is important to count nuclei accurately for single-cell analysis. Dual fluorescence counting using the CellDrop™ FL Automated Cell Counter can clearly distinguish nuclei, unlysed intact cells and debris by acridine orange/propidium iodide (AO/PI) counting.

Technical Note highlights:

    • It describes how the CellDrop Automated Cell Counter uses dual fluorescence to distinguish between isolated nuclei, unlysed cells and debris to provide accurate counts for single nuclear RNA-Sequencing or ATAC-Seq workflows.
    • The method will label successfully isolated nuclei red and any remaining intact cells green. This allows the user to calculate the residual intact cells that carryover as a percent of total counted and determine if the experimental workflow can proceed.
    • Compares nuclei counting data using dual fluorescence with traditional trypan blue staining demonstrating significant improvements in differentiation of nuclei, cells and debris to improve library preparation.

 

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