Question: How should I count nuclei after isolating nuclei using the Chromium Nuclei Isolation Kit?
Answer: 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.
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.
Trypan blue staining is not the ideal assessment and counting method for nuclei suspensions. Still, it can be used as an alternative if a fluorescent-capable automated counter or microscope is unavailable. Using trypan blue staining for counting nuclei suspensions can result in overcounting of nuclei as debris can stain with Trypan Blue and be included in the nuclei counts.
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