How do I know if my cell sample is good enough for CUT&RUN?
As outlined in this article, EpiCypher checks sample integrity at three steps during the CUT&RUN protocol. It is crucial that samples pass all three sample checks, as poor quality samples increase assay background and reduce signal-to-noise.
EpiCypher focuses on several features when it comes to assessing cell quality: morphology/integrity, viability, and total cell counts. Ask yourself these key questions when analyzing sample quality:
- What is the normal morphology for my cell type? EpiCypher routinely works with K562 cells, which have rounded cell morphology. It is key that the cells appear normal before attaching to ConA beads.
- What is the optimal viability for my cell type? For freshly harvested, native K562 cells, EpiCypher typically observes >90% viability using Trypan Blue staining. However, viability may vary drastically by cell type or experimental conditions, such as drug treatments. Furthermore, some cells display reduced viability in CUT&RUN Wash Buffer. Be sure to follow our instructions for assessing sample quality and make the best decision based on your sample type.
- Do I have the appropriate number of cells? EpiCypher recommends using 500,000 cells per CUT&RUN reaction. Count cells both at time of harvest and before ConA bead binding. The second count will confirm you have not lost significant sample during prep and ensure confirm cell morphology before proceeding with the experiment.
- Are my cells being accurately counted and assessed? Trypan Blue is toxic to cells, and some cells may display high sensitivity. As such, it is important that Trypan Blue dye is added immediately before cell counting. If your cells show low viability, yet have normal morphology, minimal debris/lysis, and have been successfully expanded in culture, it may be prudent to test a different cell counting method. Propidium Iodide is a similar nuclear dye that has been implemented with success in several collaborator labs. Alternatively, Trypan Blue can be used at a lower dilution.