Question: What should I do if I have >5% of my nuclei suspension staining live?
Answer: We recommend having <5% live cells in the final nuclei suspension. If you have >5% of cells labeled as live, it may be an issue with the live/dead staining or the cell counter. If using trypan blue, the staining may be inefficient and some nuclei only weakly take up the stain, making them appear viable. Myelin debris (and other cellular debris) can also sometimes take up the trypan blue stain, causing an over-estimation of cell counts. If using a fluorescent dye (like AO/PI) with an automatic cell counter, the reported viability may be off due to the cell counter settings.
To determine nuclei suspension concentration, viability, and quality after isolation, visualize nuclei suspensions at 40x or 60x magnification using a nucleic acid staining fluorescent dye (e.g., Ethidium Homodimer-1 or Propidium Iodine) and a fluorescent capable automated counter or microscope. Using a fluorescent dye improves nuclei counting accuracy as nuclei counts will not include debris. We recommend visually inspecting the cell counter field of view to ensure cells are appropriately being called as live or dead and nuclei are accurately stained for nuclei acid content.
When using trypan blue, live cells should have distinct dark borders with light centers while dead cells should appear uniformly dark. If sufficient focus or lighting is not achieved, reported viability may be inaccurate. Using the auto-focus and auto-light adjustment can sometimes resolve these issues. Small cells and isolated nuclei (<10 µm) may be difficult to distinguish from debris or other particles and some cell counters (such as the Countess 3 FL) have gating parameters that may not be set to accurately detect very small nuclei. You can check to see if your automatic counter has adjustable cell gating parameters (settings 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 and a fluorescence-based nucleic acid specific dye 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 and as a result may give inaccurate nuclei counts.
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