Fig. 2

Cone photoreceptors can be easily localised based on their EGFP expression and their scatter characteristics. The figure shows the gating strategy used for the analysis and sorting of the EGFP positive cells. The first selection was only based on the scatter characteristics of cone photoreceptors (forward versus side scatter a, b) Flow cytometry cell sorting was performed using an endogenous EGFP transgene expression to enrich for cone photoreceptors with a threshold filter that removes autofluorescent cells. A drop of DRAQ7 DROP AND GO™ rapid dsDNA-dye was added to identify dead cells (excited with 633 nm laser, emission collected with the 675/25 nm Accuri C6, 665/25 nm in the Cyan ADP and the 660/20 nm in the FACSAria IIIu) which were discarded. Dead EGFP⁺ cells were discarded as they are positively labeled for DRAQ7. The gate EGFP⁺ DRAQ7⁻ (c, d) corresponds to all the EGFP positive, viable cells. The gate sorted populations is used to minimise any possible EGFP-contaminations due to autofluorescence. Table (d) represents one representative sorting process, and lists cell number and percentage of gated cells which were identified in the EGFP⁺ DRAQ7⁻ and the final sorted population