How to accurately count cell nuclei in single-cell nuclear sequencing?

Single-cell genomics technologies are currently in full swing, such as single-cell RNA sequencing (scRNA-seq) and chromatin transposase accessibility sequencing (ATAC-seq)

In this process, we must accurately count the nuclei

Compared with traditional large-volume cell sequencing methods, single-cell genomics technology highlights the differences between cells and helps to gain a deeper understanding of sample materials

However, such studies require accurate counting of isolated nuclei, because the requirements for library preparation are the minimum number of unlysed cells, and the sample does not contain cell clumps and debris

AO/PI staining is more reliable than trypan blue

People usually use cell viability indicators to count the isolated nuclei

For this reason, acridine orange/propidium iodide (AO/PI) staining is the first choice, whether for routine cell viability testing or single-cell genomics research

This method has also recently been applied to single-cell genomics applications, where green corresponds to intact cells and red corresponds to successfully isolated nuclei

Fully automated cell counting

For samples stained with AO/PI, we can manually evaluate the staining results under a fluorescence microscope

At the same time, when selecting an automatic cell counter for single-cell genomics research, in addition to the fluorescence counting capability, the need for the sample size of the instrument should also be considered

In addition, when dealing with challenging samples, such as tissue lysates, the advantages of AO/PI staining over trypan blue are more obvious

Researchers have used an automatic cell counter to count cell nuclei isolated from mouse brains and compared the two methods

There are many fluorescence cell counters on the market, such as the CellDrop fluorescence/bright field cell counter