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. As mentioned in our Demonstrated Protocol for the Isolation of Nuclei, we have used PI to count nuclei.
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 a negative dye like DAPI, Sytox, or PI which does 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.
If using a live-cell permeant dye like Hoechst, and collecting cells via positive selection, then it is most likely fine, but this dye is not specifically tested so proceed with caution.
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. Again, Calcein AM is untested. 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.
If using a live-cell permeant dye like Hoechst, and collecting cells via positive selection, then it is most likely fine, but this dye is not specifically tested so proceed with caution.
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. Again, Calcein AM is untested. 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.
A dye like AO enters both live and dead cells and binds to DNA, and should be avoided as it hasn't been specifically tested.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome