Question: After CMO-labeling and pooling, how long can cells or nuclei sit before loading onto the chip?
Answer: It is critical to work efficiently and avoid letting cells/nuclei sit for extended periods, as this can decrease cell viability or nuclei quality, and can lead to increased background in 3’ CellPlex data.
- If CMO-labeled cell or nuclei samples are pooled together and left to sit for extended periods of time, CMO tags from one sample may bind to cells/nuclei from another sample, increasing background noise in the Cell Multiplexing data and decrease the accuracy of the tag assignment algorithm.
- Pool cells/nuclei as soon as possible after CMO labeling and washing (within 30 minutes). After pooling, load cells/nuclei onto the 10x Genomics chip as soon as possible (within 30 minutes).
- Keep cells/nuclei on ice after each wash step and after completion of the CMO labeling and washing protocol. Do not let CMO labeled cells/nuclei sit at room temperature, as this may increase background in 3’ CellPlex data.
Note for customers interested in performing flow cytometry:
We have performed in-house experiments where we pooled CMO-labeled cells together and performed flow cytometry on the pooled sample to enrich for viable cells. We observed excellent Cell Multiplexing data with these experiments (See: Is flow cytometry compatible with the 3’ CellPlex Kit for Cell Multiplexing?). This suggests that the CMO tag was stable for the flow sorting experiment's duration. However, after flow sorting, it is important to keep the cells/nuclei on ice and load the samples onto the 10x Genomics chip as soon as possible after flow sorting.
Products: Single Cell Gene Expression, CellPlex