What are the best practices for flow sorting cells for 10x Genomics assays?

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 Genomics Single Cell experiment. Flow sorting of cells can allow you to select for a specific population of interest while also enriching for live cells and removing debris. 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. These guidelines emphasize gentle cell handling to preserve cell health and viability, which are critical to the success of a 10x Genomics experiment. 

Cell loss during flow sorting is common. Optimize the protocol steps accordingly.

Additional guidance can be found in our Flow Cytometry Technical Notes CG000720 and CG000598. 

Buffers

• Pre-sorting buffer
  • When preparing the single cell suspension that will be used for flow sorting, we recommend using serum rich buffers to maintain cell viability. The optimal serum concentration may vary based on the sample type. In-house we have tested the following buffers to suspend the cells in prior to sorting:
    • PBS + 10% FBS was tested in our Cell Surface Protein labeling protocol and Cell Multiplexing Oligo labeling protocol 
    • PBS + 2% FBS was testing in our Barcode Enabled Antigen Mapping protocol and our dCODE™ Dextramer® labeling protocol 
• Collection buffer for collecting cells post-sorting
  • Pre-coat the collection tubes by adding 1 ml PBS + 20% FBS, then removing it
  • Add an appropriate volume of collection buffer (20ul to 30ul) to the collection tube. 
    • PBS + 5% FBS for collecting <10,000 sorted cells
    • PBS + 20% FBS for collecting <500,000 sorted cells
    • 100% FBS for collecting >500,000 sorted cells
    • A higher percentage of FBS is needed to maintain cell viability when sorting larger cell numbers, as the Collection Buffer is diluted during sorting. Up to 10% FBS in the final cell suspension is compatible with downstream Single Cell 3' or 5' assays. 
    • Detailed guidance on collection buffers and volumes can be found in our Barcode Enabled Antigen Mapping (BEAM) User Guide CG000595.

Pre-sorting

• Make sure that your stream is directed into the collection buffer and is on.
• Ensure that an appropriate volume of buffer is in the collection tube(s) to cushion the cells as they land.
• Ensure that the sheath fluid and collection buffer is compatible with the 10x workflow--importantly, it does not contain EDTA or excessive amounts of Mg2+ (should be less than 0.1 mM EDTA and less than 3 mM Mg2+).
• Include a dead cell marker in the sorting scheme to exclude dead cells.

During sorting

• 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.
• Keep the collection tube chilled during sorting. 

Post-sort

• 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 (we do not recommend this unless you have >50,000 cells, given that the cell recovery may post centrifugation may be low). 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.

Considerations for sorting nuclei in addition to the above guidance:

Pre-sort:

• Ensure that an appropriate volume of buffer is in the collection tube(s) to cushion the nuclei as they land. The amount of BSA and RNase inhibitor in the collection buffer should be based on the final volume after sorting is complete rather than the initial volume prior to sorting.
  • Example calculation: The final volume after sorting will be 2ml and the final collection buffer should contain 1% BSA and 1U/ul RNase inhibitor. If the collection tube starts with 1ml buffer prior to sorting, the buffer should include 2% BSA and 2,000U RNase inhibitor so that they are diluted to the correct concentrations during sorting.
• Do not sort nuclei that have been permeabilized. Permeabilization to increase transposase access for the ATAC or Multiome assays should be conducted after sorting. Please refer to CG000375: Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing.
• Assess nuclei quality for leaking, blebbing, and/or tears in the nuclear envelope prior to sorting. The stresses associated with sorting may further deteriorate low quality nuclei.
• If you are staining nuclei, we strongly recommend 7-AAD. Alternative dyes may reduce performance of ATAC and Multiome assays (see Can I sort nuclei for Single Cell ATAC sequencing or Single Cell Multiome ATAC + GEX?).

Post-sort:

• Carefully assess nuclei quality prior to loading the 10x chip (for gene expression assays) or beginning transposition (for ATAC and Multiome assays, after nuclei are permeabilized and resuspended in Diluted Nuclei Buffer). We recommend using high magnification brightfield microscopy to inspect nuclei for leaking, blebbing, and/or tears in the nuclear envelope. Sorting can stress nuclei and decrease nuclei quality.

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