Question: Is flow cytometry compatible with the 3’ CellPlex Kit for Cell Multiplexing?
Answer: Yes, CMO-labeled cells or nuclei can be sorted using fluorescence-activated flow sorting (FACS)
After labeling cells/nuclei with Cell Multiplexing Oligos (CMOs), flow sorting samples can improve Cell Multiplexing data quality by reducing background from debris and unbound CMOs.
- CMO-labeled cells can be sorted using a fluorescent live/dead marker. Cells can also be labeled with both CMOs and fluorescent antibodies and sorted to enrich specific cell populations.
- CMO-labeled nuclei can be sorted based on forward and side-scatter and a fluorescent DNA dye such as 7-AAD.
CMO-labeled cells or nuclei can be pooled before or after flow sorting:
- CMO-labeled samples can be sorted separately and pooled together after sorting.
- Alternatively, CMO-labeled samples can be pooled together first and then sorted together as one sample.
For a demonstration of flow sorting CMO-labeled cells, please refer to our Technical Note on Cell Multiplexing.
After performing CMO-labeling and washing as outlined in our Demonstrated Protocol, CMO-labeled cells/nuclei can be suspended in an appropriate buffer for flow cytometry. For in-house experiments, CMO-labeled cells/nuclei were suspended in the following buffers:
- CMO-labeled nuclei: PBS + 1% BSA + 0.2U/ul RNase inhibitor
- CMO-labeled cells: PBS + 10% FBS
Other buffer types are likely to be compatible and we recommend selecting a buffer that allows for maximal viability of your sample type. Please refer to this article for general guidance on buffers compatible with the Single Cell Gene Expression assay.
For additional guidance, please see: What are the best practices for flow sorting cells for 10x Genomics assays?
To reduce background noise in CellPlex data, use a FACS instrument with a chilled sample chamber and chilled collection chamber, and keep samples on ice before and after sorting.
Number of wash steps after CMO labeling
If flow sorting is performed on CMO labeled cells, it is possible to perform one wash step instead of three wash steps after CMO labeling, given that the flow sorting step itself acts as a very efficient wash step. Please refer to Protocol 3 and Protocol 4 in the CMO labeling Demonstrated Protocol for guidance on reducing the number of wash steps prior to FACS.
Gating strategy for CMO-labeled samples
For some viability dyes used for FACS, a signal shift may be observed, with an overall increase in signal intensity in CMO-labeled samples as compared to unlabeled samples. This does not negatively impact FACS performance. As long as the FACS gates are set up appropriately to account for this shift, it is still possible to distinguish positive and negative cells. For example, our NSCLC dataset, which is publicly available on our Support site, was generated using FACS to isolate viable cells (7-AAD negative) after CMO labeling. In the FACS plots associated with this dataset, the 7-AAD negative and positive populations are clearly distinguishable from each other:
Products: Single Cell Gene Expression, CellPlex