Question: Which buffers can be used for the wash steps post CMO labeling when using the 3’ CellPlex kit for Cell Multiplexing?
Answer: As outlined in our Demonstrated Protocol, the 3’ CellPlex assay was validated using the following buffers to wash cells/nuclei after CMO labeling:
| Wash buffer post CMO labeling | Sample type |
| PBS + 1% BSA | PBMCs, cell lines, dissociated tumor cells |
| NbActiv-1 + 1% BSA | Dissociated brain tissue |
| PBS + 10% FBS | Cells with viability >70% but <80% |
| PBS + 1% BSA + RNase Inhibitor (0.2 U/μl) | Nuclei |
If using BSA, we do not recommend decreasing the percentage of BSA in the wash buffer below 1%. During in-house experiments, we found that using 1% BSA, compared to various other wash buffers tested (including 0.04% BSA), produces the best signal-to-noise ratios in CellPlex data and also minimizes cell/nuclei loss and aggregation.
For sensitive cell types, 10% FBS can be used instead of 1% BSA, to maximize cell viability.
For sensitive cell types, a cell culture media appropriate for the sample type can also be used instead of PBS. For example, for the dissociated brain tissue mentioned above, we used the neuronal cell culture media NbActiv-1. For general guidance on selecting cell culture media compatible with the downstream Single Cell 3’ Gene Expression assay, please refer to this article.
Note on using BSA concentrations above 1%:
We have not tested using BSA concentrations higher than 1% in the wash buffer post CMO labeling. Our recommended upper limit for BSA in the standalone Single Cell 3' Gene Expression assay is 2% BSA, but we have not specifically tested 2% BSA in the 3' CellPlex assay. Also see: What is the highest BSA concentration that can be used in the single-cell suspension buffer?
Note on buffers to use prior to CMO labeling:
For in-house experiments, cells/nuclei were suspended in the following buffers prior to CMO labeling:
| Resuspension buffer prior to CMO labeling | Sample type |
| PBS + 0.04% BSA | Cells |
| PBS + 10% FBS | Cells with viability >70% but <80% |
| PBS + 1% BSA + RNase Inhibitor (0.2 U/μl) | Nuclei |
If you use alternative cell/nuclei isolation protocols that include different resuspension buffers, we recommend washing and resuspending the cells/nuclei in our recommended buffers before CMO labeling. This will avoid introducing components that may interfere with the CMO labeling reaction. For example, if detergents are used during nuclei isolation, wash steps should be performed with PBS + 1% BSA + RNase Inhibitor to remove detergents before CMO labeling.
Products: Single Cell Gene Expression, CellPlex