Question: Can post-fixation samples be sorted in the Fixed RNA Profiling assay?
Answer: Post-fixation samples can be sorted using FACS for advanced sample clean-up, as well as for the enrichment of specific populations in the Fixed RNA Profiling Assay (i.e., Single Cell Gene Expression Flex).
Advanced sample clean-up
Post-fixation samples can be flow sorted for advanced sample clean-up to remove cellular debris from single-cell or nuclei suspensions using 7-AAD. DAPI, Hoechst, and other DNA fluorophores are also expected to be compatible with staining fixed and nucleated cells.
Debris heavy tissues such as brain may benefit from sorting to remove cellular debris after tissue fixation and dissociation following the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling
Demonstrated Protocol. However, it is important to count post-sorting before proceeding with probe hybridization, given that the cell recovery will be reduced.
Enrichment of specific populations:
Fixation is not expected to preclude FACS isolation for most fluorophores. Some fluorophores may be impacted by longer fixation. We have performed limited testing for the enrichment of specific populations post-fixation (1 hr & 24 hr fixation) using FACS. Cells should be labeled prior to fixation following the Cell Surface Protein Labeling for Chromium Fixed RNA Profiling for Singleplexed Samples with Feature Barcode technology Demonstrated Protocol. Following cell surface protein labeling, the cells should be immediately fixed following the guidance in the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling Demonstrated Protocol.
Limited testing for the enrichment of specific populations post-fixation using FACS found that although slight shifts in the ssc and fsc profiles were observed, both positive and negative populations could be distinguished in samples that were sorted post-fixation. Fluorescent reporter proteins such as EGFP and RFP may be dimmer after fixation, but -/+ populations could still be distinguished in samples that were sorted post-fixation. Additional optimization may be required for sorting samples post-fixation. A pilot experiment is recommended prior to committing to a larger study.
Fluorophores that were found to be incompatible with fixation (limited testing):
- FITC
- Brilliant Violet 605™
- PE-Cy7 (tandem dye)
- PE-Dazzel 594 (tandem dye)
Below is a demonstration for sorting labeled fixed cells. Human PBMCs were stained them with two fluorescent antibodies targeting T cells and B Cells following the Cell Surface Protein Labeling for Chromium Fixed RNA Profiling for Singleplexed Samples with Feature Barcode technology Demonstrated Protocol, and then fixed for either 1hr at room temperature, or overnight at 4C. After fixation, a portion of these cells and sorted for CD3 positive cells or CD19 positive cells. These sorted samples were then used in the Flex assay. The non-sorted sample gave comparable data to the sample sorted for all populations, suggesting that flow sorting does not seem to impact Flex data quality. The purity of each cell type, as well as the representative gene expression, for both 1 hr fixed and overnight fixed samples for the individual sorted T cells and B cells is shown on the right.
Best practices for sorting fixed samples:
- Samples should be collected in PBS + 1% nuclease- free BSA supplemented with RNase inhibitor (Protector RNase inhibitor from Sigma, PN-3335399001). We recommend a final concentration of 0.2 U/ul of RNAse inhibitor.
- Samples should be spun down and resuspended in PBS + 0.02% nuclease-free BSA before proceeding with probe hybridization.
It is always recommended to consult a core facility or an experienced flow user before initiating a flow sorting experiment.
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex