Unbiased method for spectral analysis of cells with great diversity of autofluorescence spectra

The purpose of this manuscript is to provide, visualize and verify an unbiased workflow for spectral flow cytometry to identify and select all unique autofluorescence clusters within heterogeneous autofluorescence samples, which can be used during the unmixing to improve signal-to-noise ratios.
Conclusion:
Using the proposed unbiased workflow, the unmixing of heterogeneous autofluoresence samples acquired using spectral flow cytometry, is improved. Moreover, this method identifies all different unique autofluorescence spectra, which can be used for other purposes, like autofluorescence-based sorting of cells. Sample acquisition was done in all cases after successful instrument embedded QC procedures as recommended by the manufacturer. For appropriate autofluorescence spectra detection and unmixing, an unstained sample was taken along during the procedure, that was treated according to exactly the same procedure as the stained sample, of which at least the same amount of cells as in the stained sample were acquired. UltraComp eBeads compensation beads (ThermoFisher) were used as single stain controls for all fluorochrome-labelled antibodies, and cells heat shocked for 3 minutes at 65 °C were taken along as single stain control for the viability dye.