Fluorescence-Activated Cell Sorting (FACS) isolated cells or nuclei can be successfully used for CUT&RUN. However, the FACS process can result in cell loss and increased cell fragility, and special care should be taken when handling these samples. Read on for our latest recommendations and answers to common questions.
FAQ: Do intracellular stains impact CUT&RUN profiling?
Intracellular stains for nuclear proteins, such as transcription factors, can interfere with downstream CUT&RUN. These antibodies also interact with pAG-MNase, contaminating CUT&RUN sequencing data.
For this reason, we do NOT recommend using a nuclear antibody for FACS when performing downstream CUT&RUN. If you must use an antibody for sorting, we recommend one that targets a cell surface protein.
Fix samples to help maintain cell integrity during FACS
FACS is a stressful process for live cells and can make them more prone to lysis during CUT&RUN. Light fixation prior to FACS (i.e. 2% formaldehyde) can help stabilize cells and is standard procedure in many sorting facilities.
Note that cells should be stained prior to fixation. Additionally, the sorting dye must be one that can be fixed, such as the Thermo Fisher Live/Dead Fixable Stain.
Count FACS isolated cells (or nuclei) prior to starting CUT&RUN
Depending on the type of sample you are isolating and starting number of cells, your final cell number may be quite low.
Although the FACS machine reports a final cell count, we have found that this number is unreliable, and may be several orders of magnitude higher or lower than the true number of cells in your final sorted populations.
For this reason, it is crucial to manually recount cells after sorting and prior to beginning CUT&RUN. Keeping the density between 1-5 M cells/mL will help maximize the cell counter’s accuracy.
Considerations when using FACS-nuclei for CUT&RUN
Nuclei can be extracted before or after FACS and used as the input for CUT&RUN. However, extracting nuclei after FACS is preferable, as nuclei can be fragile and so may be less likely to survive the lengthy sorting process.