Epics elite esp cytometer

Flow cytometry was used to determine the DNA content of sesame [54 ]. Sesame samples and reference material were analyzed on an EPICS Elite ESP cytometer (Beckman-Coulter, Hialeah, FL, USA) with an air-cooled argon laser (Uniphase) at 488 nm using 20 mW. Salmon erythrocytes (2.16 pg/1C) were used as internal biological reference materials. Nuclear DNA content (in picograms) of sesame samples was estimated according to the following equation: 1C nuclear DNA content = (1C reference in picograms × Peak mean of sesame)/(Peak mean of reference). The number of base pairs per haploid genome was calculated based on the equivalent of 1 pg DNA = 978 Mb [55 (link)]. As a result, the C-value of sesame was estimated to be 0.34 pg/1C, and its genome size was estimated to be approximately 337 Mb (Figure S3 in Additional file 1).

The pineal gland and the retina were dissected from adult fish of the Tg(exorh:egfp) and Tg(rho:egfp) transgenic lines, respectively. These tissues were digested in 0.25% trypsin/1 mM EDTA in phosphate-buffered saline (0.8% NaCl, 0.02% KCl, 20 mM phosphate, pH 7.3) at 37 °C for 30 min, and then the reaction was stopped by addition of an equal volume of phosphate-buffered saline containing 2% (w/v) soybean trypsin inhibitor and 20% (v/v) fetal bovine serum. After filtration through a 35-µm nylon mesh, the dissociated cells were sorted by using EPICS ELITE ESP cytometer equipped with Expo32 software (Beckman Coulter). Forward scatter, side scatter and 525-nm green fluorescence intensity were monitored for gating. Sorted cells were directly collected into 1.5-ml microtubes containing TRIzol reagent (Invitrogen) for subsequent extraction of total RNA.