We have developed a male-specific, fluorescent stain that can be used in conjunction with the LPD 1000NF (Union Biometrica, Inc.) to purify male worms from a mixed population. The LPD 1000NF is a high throughput system that analyzes and sorts C. elegans using parameters of size and fluorescence. The stain is comprised of a specific ligand to the male copulatory bursa. The ligand is coupled to a dye that is excited by a standard 633nm HeNe laser and emits in the far red spectra. A
him-8 strain, CB1489, was used in order to provide sufficient males for development of the reagents used in this study. Mixed populations treated with the stain were analyzed and sorted on a LPD 1000NF system. A sort region was set on a size versus fluorescence two-parameter display and the brightest worms were dispensed. The sorted males were successfully used in mating studies and the stain in no way inhibited copulation. The sorted male worms were mated with a strain which has high-level GFP expression in the pharynx, in order to demonstrate that the stain did not inhibit copulation. The progeny were examined using the LPD 1000NF system. Three populations were observed on size versus fluorescence, two-parameter plots. One population had a fluorescence level equal to that of the GFP expressing strain; a second population showed no GFP expression, indicating that mating had indeed occurred; and the third population showed an intermediate level of fluorescence. The intermediate population consisted of heterozygous worms that had only one chromosomal copy of GFP.