Fluorescent dna quantitation kit

Cell proliferation assays were performed as previously described.^{49 (link)} The DNA content of the cells was determined using a Fluorescent DNA Quantitation kit (Bio-Rad Laboratories, Hercules, CA). For each analysis, three replicate wells were used, and at least three independent experiments were performed.

Nasal wash samples were collected at 1 to 16 h after intranasal administration of liposomes (400 nmol/mouse) by flushing with 100 µL of cold PBS. After centrifugation to remove cells/debris, the amounts of dsDNA in the nasal wash samples were measured by a Fluorescent DNA Quantitation Kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions using a Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments, Inc.).

Total cellular triglyceride levels in RIPKO-1 and RIPKO-L cells were determined with the triglyceride colorimetric assay kit (Cayman Chemical) according to the manufacturer's instructions. Data was normalized to DNA content measured using fluorescent DNA quantitation kit (Bio-Rad Laboratories).

Total DNA was extracted directly from musts and wines using the PowerSoil DNA Isolation Kit (MoBio Laboratories). Ten milliliter of each sample were centrifuged to collect cells. The DNA was then extracted according to manufacturer's protocol. Quantification of total DNA was achieved using a VersaFluor fluorimeter and a Fluorescent DNA Quantitation Kit (Bio-Rad, Milan Italy). DNA was used as a template for microsatellite PCR fingerprinting, as described by Vaudano and Garcia-Moruno (2008 (link)). PCR amplifications were performed in a thermocycler (MyCycler, Bio-Rad Laboatories, Milan, Italy) with the following PCR programme: 4 min of initial denaturation at 94°C, 28 cycles of 30 s at 94°C, 45 s at 56°C, 30 s at 72°C and, finally, 10 min at 72°C. The products were run on a 2.5% (w/v) agarose gel 1 × TAE buffer at 100 V for 80 min. Gels were stained with ethidium bromide. 1-kb plus DNA ladder (Life Technologies, Milan, Italy) was used as marker for the gel normalization.

Chondro-pellet cultures (0.25 × 10⁶ MSC) were induced towards chondrogenesis for 21 days with serum-free MesenCult-ACF differentiation medium (STEMCELL Technologies Inc, Vancouver, Canada). Harvested pellets were cryosectioned and 6-μm frozen sections stained with 1% toluidine blue (Sigma). Sulfated glycosaminoglycans (sGAG) were quantified after pellet digestion at 65 °C overnight with 1 mg/ml papain (Sigma) solution using the Blyscan Glycosaminoglycan Assay (Biocolor, Carrickfergus, UK) according to manufacturer’s instructions. DNA content was quantified using Fluorescent DNA Quantitation Kit (Bio-Rad Laboratories, Hercules, CA) according to manufacturer’s instructions.