Dna quantitation kit

After 21 days, pellets were digested using papain, and total amount of GAG glycosaminoglycans content was assessed using chondroitin-6-sulphate DMMB (1, 9 Dimethyl methylene blue) method. DMMB absorbance was read using a microplate reader. Total GAG amount per pellet was normalized to DNA content per pellet. The total DNA content was obtained using a DNA Quantitation Kit (Sigma Aldrich, Burlington, MA, USA), in which bisbenzimide, a fluorescent dye, binds with double stranded DNA so that when excited at 360 nm, the fluorescence emission at 460 nm increases proportionally to the amounts of DNA from the sample. The results are estimated using a standard curve.

Double-stranded deoxyribonucleic acid (dsDNA) and major ECM components were quantified from decellularized (DT) and compared with native (NT) LV mouse heart tissue. The dsDNA content was determined using the DNA Quantitation Kit (Sigma-Aldrich, DNAQF) according to the manufacturer’s instructions. The quantity of protein, collagen, and sulfated glycosaminoglycans (sGAG) was quantified using BCA assay (Thermo Scientific, PI23225), QuickZyme Total Collagen assay (Quickzyme Biosciences, QZBTOTCOL1), and Glycosaminoglycan assay Blyscan (Biocolor, B1000), respectively, according to the manufacturer’s instructions. Data were expressed as measured component mass normalized to tissue wet weight or protein content.

After 7, 14 or 21 days of incubation, each cell−hydrogel construct was removed from the 24-well plates and washed twice with PBS, incubated with 500 µl lysis buffer (0.25% Triton X-100 in 0.5 M 2-amino-2-methyl-1-propanol). After vortexing, the extracted mixtures were centrifuged and the supernatants were collected for calcium and DNA analysis (N = 3). Calcium contents of the encapsulated hMSCs were quantified using a calcium assay kit (Cayman Chemical). The absorbances were measured at 570 nm on a microplate reader. Calcium concentration of each sample was calculated based on the standard curve. DNA contents were also analyzed using a DNA Quantitation Kit (Sigma-Aldrich) and fluorescence intensities of the dye-conjugated DNA solutions were measured using a fluorescence spectrophotometer (Horiba Scientific). All calcium contents were normalized by DNA contents.

The DNA Quantitation Kit (Sigma-Aldrich Cat. no. DNAQF) was used to determine the amount of DNA present in the experimental samples. The assay is based on fluorescent dye, bisBenzimide H 33258 (Hoechst 33258), which binds primarily to AT sequences in the minor groove of double-stranded DNA (dsDNA). The assay was set up in a 96-well plate format and the readouts were measured using a multi-mode microplate reader FLUOStar Omega (BMG Labtech, Ortenberg-Germany). Fluorescence excitation and emission wavelengths were set to 355 nm and 460 nm respectively. Average results from 5 cycles with 10 individual measurements per cycle have been used to calculate the DNA content of the sample.

For the quantitative measurement of cell proliferation, SF specimens were harvested and digested at 60 °C for 24 h with papain solution (50 μg/mL with 55 mM sodium citrate, 150 mM sodium chloride, 5 nM cysteine HCl and 5 mM EDTA (ethylenediaminetetraacetic acid)). Total DNA content was measured by a DNA Quantitation Kit (Sigma-Aldrich) with calf thymus DNA as the standard. The digested solution was also used to quantify the amount of sulfated GAG present in the specimen. The amount of GAG was quantified using 1,9-dimethylmethylene blue reagent. Shark chondroitin sulfate was used as a standard to detect the GAG content, and measurements were recorded at 525 nm by a spectrophotometer. For the quantification of collagen Type II (Col II) content, the specimens were digested in 1% (w/v) pepsin in 0.05 M acetic acid and further solubilized with 0.1% (w/v) pancreatic elastase solution in Tris buffer saline (pH 8.0) at 4 °C for 24 h. The concentration of Col II was determined with a Type II collagen ELISA kit (MD Bioproducts) at 490 nm.