Calf thymus dna

All commercially available chemicals were reagent‐grade and used without further purification unless otherwise mentioned. Spectroscopic grade solvents were used for solutions submitted to absorption and emission spectroscopy. All buffer solutions were prepared from purified water (resistivity 18 MΩ cm) and biochemistry‐grade chemicals. The buffer solutions were filtered through a PVDF membrane filter (pore size 0.45 μm) prior to use. The oligodeoxyribonucleotide 22AG d[A(GGGTTA)₃GGG] (purification: HPLC; quality control: MALDI‐TOF; synthesis scale: 1.0 μmol) was purchased from biomers.net GmbH (Ulm, Germany). Calf thymus DNA (type I; highly polymerized sodium salt; ϵ=12824 cm⁻¹ 
m⁻¹) was purchased from Sigma–Aldrich. Poly(styrene sulfonic acid) (PSS, sodium salt, M.W. 70 000) was purchased from Alfa Aesar. BPE (biphosphate EDTA) buffer: 6.0 mm Na₂HPO₄, 2.0 mm NaH₂PO₄, 1.0 mm Na₂EDTA, pH 7.0; potassium phosphate buffer: 25 mm K₂HPO₄, 70 mm KCl; adjusted with 25 mm KH₂PO₄ to pH 7.0.

Kunitz assays were performed with 1 pmol/μl of purified recombinant hPLSCR1, 50 μg/ml of calf thymus DNA (Sigma Aldrich, USA) as the substrate in a buffer containing 50 mM Tris–HCl (pH - 8.0), 150 mM NaCl, 10 mM MgCl₂ and 0.5 mM dithiothreitol to a total volume of 500 μl and incubated at 37 °C for 15 min. Negative controls contained only the DNA substrate without hPLSCR1. The difference in A₂₆₀ between control and sample was considered for measuring the nuclease activity. One Kunitz unit is defined as the amount of enzyme added to 1 mg/ml of DNA that causes an increase in absorbance of 0.001 per minute at 260 nm at 37 °C. The increase in absorbance is due to the release of free nucleotides upon degradation of polymerized DNA [35 (link)]. Briefly, the enzyme activity is quantified as follows.

Amount of DNA : 25 μg (final concentration – 50 μg/ml)

Dilution factor : 20

Amount of protein : 1 pmol/μl

Incubation time : 15 min

Test : DNA treated with hPLSCR1

Control : Untreated DNA

$\mathrm{Enzyme}\mathrm{activity}=\frac{\left[A_{260}\left(\mathit{test}\right)-A_{260}\left(\mathit{control}\right)\right]\times 20}{0.001\times 15}$

Azocasein, phenyl methyl-sulfonyl fluoride (PMSF), tosyl phenylalanyl chloromethyl ketone (TPCK), tosyl lysine chloromethylketone (TLCK), E-64 (10 μM), ethylenediaminetetraacetic acid (EDTA), N-α-benzoyl-Arg p-nitroanilide (BApNA), N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA), leucine p-nitroanilide (LpNA), p-Glu-Phe-Leu-pNA (pGPLpNA), Z-Arg-Arg-pNA (AApNA), calf thymus DNA and baker’s yeast RNA were purchased from Sigma-Aldrich, St. Louis, MO, (USA).

Preparation of standard DNA containing AP sites was performed according to Lindahl and Nyberg's procedure [3 (link)]. Calf thymus DNA (Sigma–Aldrich) was dissolved in TE buffer and purified by phenol-chloroform extraction and ethanol precipitation. The purified DNA was treated with 100 μg/ml RNase A (Sigma) at 37°C for 1 h and then subject to phenol–chloroform extraction and ethanol precipitation. The DNA solution was dialyzed against AP buffer (10 mM sodium citrate, 100 mM NaCl, pH 5.0) and diluted at 1 mg/ml; dialyzed DNA was incubated at 70°C for 0–50 min and recovered by ethanol precipitation; this procedure is reported to form 1 AP site/10⁴ nucleotides/9.26 min [3 (link)]. The numbers of AP sites and methylated bases in MMS-treated and untreated cells were calculated based on the absorbance of this standard DNA.

To evaluate the cell removal and preservation of ECM components in the RdECM, biochemical assays were conducted. For the assessment of DNA, glycosaminoglycans (GAGs), collagen, lyophilized native tissues, and the RdECM were digested using papain (125 μg/mL) in 0.1 M sodium phosphate with 5 mM Na2-EDTA and 5 mM cysteine-HCL at pH 6.5 for 17 h at 60 °C. As a diluent, papain solution without tissue samples was used. The DNA content was quantified using a DNA quantitation kit (DNAQF, Sigma), according to the manufacturer’s protocol. The fluorescence intensity was measured using a fluorescence spectrophotometer (Gemini XPS Microplate Reader, Molecular Device; excitation wavelength: 350 nm, emission wavelength: 460 nm). Calf thymus DNA (Sigma-Aldrich, St. Louis, MO, USA) was used to generate a standard curve for DNA. In the analysis of GAGs, the tissue-digested solution had been stained with 1,9-dimethyl methylene blue, and the absorbance at 525 nm was read. The standard curve used for estimating the number of sulfated GAGs in the samples was plotted using chondroitin sulfate A (Sigma-Aldrich). Collagen content was determined using a total collagen assay kit (Biovision, Milpitas, CA, USA). The samples were prepared according to the manufacturer’s instructions, following measurement of the absorbance at 540 nm; hydroxyproline was used to prepare the standard curve.