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?
Achieving accurate focus and light intensity is critical to accurately counting cells and calculating viability. Live cells should have distinct dark borders with light centers while dead cells should appear uniformly dark if using a stain like trypan blue. If sufficient focus is not achieved, reported viability may be inaccurate. Using an automated cell counter that can perform autofocus and auto-light adjustment will ensure accurate cell calling and consistency between users, samples, and over time.
While the ability to manually adjust brightness and focus is useful, users should not blindly rely on automatic settings to be correct 100% of the time. Challenging samples that contain high amounts of debris, or are small in size like isolated nuclei, may benefit from an experienced user performing a visual quality control check. To ensure cells are appropriately being called as live or dead, manual adjustments of focus and lighting may help differentiate between live and dead cells in these challenging cases.
Automated counters offer additional advantages to allow for automated determination or gating of live vs dead cells. Users should familiarize themselves with these settings for their automated counter, as custom creation of cell calling protocols offer greater accuracy and consistency. Adjustable cell gating parameters may include: cell size, circularity/roundness, and brightness or fluorescent intensity.
The Cellaca MX FL5 and Luna-FL are instruments that support counting isolated nuclei. Isolated nuclei have also been counted using Countess II FL and NucleoCounter NC-202 with success. Manual counting using hemocytometer is also recommended. Countess 3 FL is currently incompatible with isolated nuclei counting due to an updated counting algorithm which is more aggressive at gating debris, defined as particles <4uM.
Products: Single Cell ATAC, Single Cell Gene Expression, Single Cell Multiome ATAC+GEX