Question: I'm having trouble accurately counting nuclei. What can I do?
Answer: Nuclei can be tricky to count due to their small size, which can be further exacerbated if a lot of cellular debris is present. The use of a nucleic acid staining fluorescent dye may help improve nuclei counting accuracy as only nucleic acids will be stained and not debris. Note that the use of a fluorescent dye requires an automated counter with fluorescent capabilities or a fluorescent microscope.
Recommended dyes for counting:
- Ethidium homodimer-1: Only nuclei are stained. Live cells and debris are unstained
- AO/PI: Acridine orange (AO) stain all cells; propidium iodide (PI) stains dead cells (i.e., nuclei), cell debris should not be stained
- It may be possible to use other nuclear staining dyes for counting
Note that some nucleic staining dyes, may affect chromatin structure and are not recommended for ATAC assays. See: Can I sort nuclei for Single Cell ATAC sequencing?
Products: Single Cell ATAC, Single Cell Gene Expression, Single Cell Multiome ATAC+GEX