Quant ittm picogreendsdna reagent

After washing with PBS to remove any unbound cells, scaffold cultures were placed directly into 1 mL lysis buffer [10 mM Tris pH 8, 1 mM EDTA and 0.2% v/v Triton X-100] within 1.7 mL microtubes. Samples were vortexed for 10 s every 5 min, for a total period of 30 min, and kept on ice between mixes. At this point, samples were either placed in -80 °C for storage or processed further. Samples were then homogenised 10-15 times using a 21gauge needle to produce cell lysate. Lysate was then diluted 1:10 in 1 × TE buffer (Thermo Fisher Scientific). Quant-iT TM PicoGreen® dsDNA reagent (Thermo Fisher Scientific) was diluted 1:200 in 1 × TE buffer. A 1:1 mixture of lysate solution and 1 × PicoGreen® reagent was placed in an OptiPlate-96, black opaque 96-well microplate (PerkinElmer). Fluorescence was measured at excitation and emission wavelengths of 460 nm and 540 nm, respectively, using an EnSpire TM 2300 Multilabel Plate Reader (Perkin Elmer) running EnSpire 3.0 software (Perkin Elmer).
Results were compared to a standard curve for 1-10 × 10 6 hPSC -NPCs analysed as above for dsDNA amount. Human PSC-NPCs cultured on laminin-coated 2D TCPS were harvested via 10 min incubation in a solution of 500 μL Accutase. Human PSC-NPCs were quantified using the trypan blue exclusion method with a 0.4% w/v solution of trypan blue and counted using a hemocytometer.