Question: What are the best practices for flow sorting cells for 10x Genomics assays?
Answer: Cell sorting is a powerful tool and can be a great way to prepare your samples for a 10x experiment. Flow sorting of cells can allow you to select for a specific population of interest while also helping to get your sample into a single cell suspension. As sorting is an advanced sample clean up methodology, it is always recommended to consult a core facility or an experienced flow user before initiating a flow sorting experiment.
Below are some general guidelines for consideration when optimizing sorting conditions before running a 10x Genomics single-cell RNA-seq experiment. These guidelines emphasize gentle cell handling to preserve cell health and viability, which are critical to the success of a 10x experiment.
Cell loss during FACS is common. Optimize the protocol steps accordingly.
- Ensure that an appropriate volume of buffer is in the collection tube(s) to cushion the cells as they land.
- Make sure that your stream is directed into the collection buffer and is on.
- Ensure that the sheath fluid and collection buffer is compatible with the 10x workflow--importantly, it does not contain EDTA or excessive amounts of MgCl2+ (should be less than 0.1 mM EDTA and less than 3 mM MgCl2+).
- Include a dead cell marker in the sorting scheme to exclude all dead cells.
- We recommend sorting based on purity as opposed to yield.
- Use a larger flow nozzle (such as a 100um nozzle, if using a sorter with a nozzle), or use lower pressure when sorting your cells. Using lower pressure during the sorting process will help preserve cell health and viability.
- In most circumstances, it is better to sort on a lower flow rate to maintain cell health. If your cell population of interest is very low (<0.01%) or sorting a lot of cells, the flow rate may need to be increased so that the sorted cells do not sit for an extended period of time before running the assay. Cells will tend to be more fragile after sorting, and sitting for a long period of time after sorting may lead to a decrease in viability.
- The higher the sorting efficiency, the better the outcome.
- Always count your cells after sorting. Counts from cell sorters tend to be inaccurate and highly variable depending on the sorter, so we always recommend recounting before loading onto the 10x chip.
- Where possible, we recommend sorting into a low volume and moving directly into GEM generation. If necessary, the collected cells may be concentrated by centrifugation and removing the supernatant. Select your spin speed according to what works best for your cell type. For fragile cells, a longer, slower spin may be preferred.
- Post-sort, move quickly but gently to get the cells into GEM generation. Sorting tends to be stressful for cells, so you want to minimize time spent on ice at this point.
- When in doubt, we always recommend consulting a flow core or other flow expert for additional guidance on maintaining cell viability.
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