Quanti it picogreen dsdna kit

The DNA content of the cells was determined using the Quanti-iT PicoGreen dsDNA kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. The cells were pre-digested in a lysis buffer (Phosphate Buffer Saline, 0.1% Triton-X 100) over night at 60 °C, sonificated and analyzed by mixing 20 µL of the digested sample with 80 µL TE buffer (200 mM Tris-HCl, 20 mM EDTA) and 100 µL PicoGreen solution. The standards were prepared from λ-DNA and DNA content was determined by fluorescence measurement at 485/535 nm.

Proteoglycan content in the cartilaginous tissue was measured by DMMB (dimethyl-methylene blue) assay, as described before [42 (link)]. For this, pellets (N = 2 per donor) were harvested at day 28 of the chondrogenic induction, washed with PBS, and digested overnight in 1 ml of lysis buffer containing 50 mM Tris-HCl (pH 8.0), and 1 mM CaCl₂ with 500 μg/ml Proteinase K (Roche, Mannheim, Germany) at 60 °C. Pellets digests (30 μl) were mixed with 200 μl of DMMB solution (38 μM dimethyl-methylene blue, Sigma, 40 mM glycine, 40 mM NaCl). Proteoglycan content was measured by spectrophotometry at 540 nm and quantified using a standard curve built with chondroitin sulphate substrate standard. The values were normalized to DNA amount in lysed cells measured using Quanti-iT PicoGreen dsDNA kit (Invitrogen, Eugene, USA). For this, 20 μl of the digested pellet sample were mixed with 80 μl TE buffer (200 mM Tris-HCl (pH 7.4), 20 mM EDTA) and PicoGreen solution, and fluorescence in samples was measured at 485/535 nm.

After treatment, 25 handpicked islets were washed 2 times with 2.75 mM glucose solution prepared in 25 mM KRH buffer (low glucose solution). After a 90 minutes preincubation period (human islets) or 30 minutes (rat islets) with low glucose solution, islets were incubated for 60 min (human islets) or 45 min (rat islets) with low glucose solution, followed by a 60 min (human islets) or 45 min (rat islets) incubation with a 16.5 mM glucose solution prepared in 25 mM KRH buffer (high glucose solution). Finally, islets were washed a single time with low glucose solution and incubated for 60 min (human islets) or 45 min (rat islets) with low glucose solution. From each incubation step, supernatants were collected, and insulin secretion was determined using the insulin ELISA kit Mercodia for human islets or Crystal Chem for rat islets. Results were normalized to total DNA content as was determined using the Quanti-iT PicoGreen dsDNA kit (Invitrogen). The stimulation index (SI) was calculated by dividing the amount of insulin secreted after incubation with the high glucose solution by the amount secreted after the first incubation with low glucose solution.