Purification of sperm and vegetative nuclei by FACS. (A) Four distinct cell populations are highlighted in the filtrate pre-sorting. Sperm cells (SC) nuclei are identified based on their strong GFP signal (Population A), whereas vegetative nuclei (VN) separate towards the opposite axis based on strong RFP signal (Population B). In addition the filtrate contains empty (Population C) and intact pollen grains (Population D). (B) Purity of sorted SC and VN fractions was confirmed by DIC and fluorescence microcopy; scale bar: 10 μm. Populations C and D were confirmed to represent empty and intact pollen, respectively; scale bar: 30 μm (C) Sorted SC and VN samples were stained with DAPI and run through a flow cytometer to check for purity. Purity is determined by measuring the percentage of SC and VN present within the total number of DAPI positive events, corresponding to all DNA-containing particles present in the sorted sample. (D) RT-PCR analyses confirmed that each fraction is enriched for cell-specific transcripts (MGH3 for SC and VEX1 for VN), and devoid of contaminating RNAs. TUB4 was used as control.