How should nuclei look for CUT&RUN?
For nuclei preparations, >95% nuclei should be Trypan Blue positive or "dead" and unclumped. Nuclei should be round, intact, and contain minimal debris (as this could indicate lysis; see Figure 1). Use Trypan Blue staining to monitor and optimize nuclear extraction, increasing spin time if losing sample.
- Note: to best ensure success, we suggest using our nuclei prep protocol.
Figure 1. Morphology characteristic of intact K562 cells (left) compared to isolated nuclei (right) when visualized under brightfield microscope after Trypan Blue staining. Isolated nuclei will stain blue, while cells will be bright white and round. For accurate nuclei counts, record "dead" cell numbers on an automated cell counter or manually count blue stained nuclei.