Question: Can cells be stained with dyes such as propidium iodide (PI) or Hoechst dyes?
Answer: In our stand-alone Single Cell Gene Expression assays, we have not tested these dyes internally. However, dyes such as PI or Hoechst have commonly used in single-cell RNA-seq projects and are not expected to interfere with the 10x Single Cell workflow. We have customers successfully use these stains for FACS upstream of the 10x Single Cell workflow.
In our Multiome assay when sorting whole cells(not nuclei), we recommend using 7-AAD (via negative selection). Dead Cells will stain 7-AAD positive and can be filtered from live cells. For whole-cell sorting, using other negative dyes like DAPI, Sytox, or PI which do not enter live cells (so unstained cells are collected), should not be a problem. However, note that we haven’t specifically tested these dyes on whole cells. Also, DAPI is known to enter live cells if the concentration is high enough. As a best practice, cells should be sorted using 7-AAD.
A dye combination such as Calcein AM with 7-AAD (or Calcein AM with ethidium homodimer -1) can also be used for sorting. Calcein will stain live cells and 7-AAD/Ethidium Homodimer will stain dead cells. Calcein- AM diffuses into cells, the 'AM' moiety is cleaved by cellular esterases, and then the dye molecules are observed in the cytoplasm without binding to anything. In theory, it should not affect your Multiome data as it is not entering the nuclei. There are some papers
reporting that the use of Calcein–based FACS to enrich viable cells may
alter the cellular composition.
Live-cell permeant dye like Hoechst or Acridine Orange(AO) may be used for collecting live cells via positive selection. However these dyes bind to DNA, so proceed with caution.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome