Normal melting point agarose

For evaluating the DNA single-strand breaks, alkaline comet assay was performed according to Singh et al. (1988) [67 (link)]. Following the treatment period, HPBL were harvested and about 3 × 10⁴ cells were embedded in a pre-heated 0.5% low melting point agarose (Sigma-Aldrich, Darmstadt, Germany) in PBS. Cells’ suspension was layered between two layers of 0.7% ultra-pure normal point melting agarose (Sigma-Aldrich, Darmstadt, Germany) on the pre-coated slides with 1% normal melting point agarose. Subsequently, slides were immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, and 10% DMSO) at 4 °C for 1 h. The slides were then incubated in the electrophoresis tank in an alkaline (pH > 13) electrophoresis buffer (300 mM NaOH and 1 mM EDTA) for 20 min to facilitate the unwinding of DNA before carrying out the electrophoresis process. After electrophoresis, the slides were neutralized with 0.4 M Tris-HCl (pH 7.5) buffer and washed with PBS then stained with ethidium bromide (20 µg/mL). Finally, 200 cells per slide were evaluated using an Olympus BX 41 UV-fluorescence microscope (Tokyo, Japan).

The SCGE assay was performed as described by Tice & Vasquez with some modifications. The remaining 20 μL of cells was placed in 150 μL of low melting point agarose (0.5%; Sigma, St. Louis, MO) and spread onto two microscope slides that had been prelayered with 180 μL of normal melting point agarose (1%; Sigma, St. Louis, MO); the slides were then immersed in a chilled lysis solution (2.5 M·NaCl, 100 mM Na₂EDTA, 10 mM Tris at pH 10, 10% DMSO, 1% Triton X-100 fresh; Sigma, St. Louis, MO). After lysis at 4 °C for 1 h, the slides were placed on a horizontal electrophoresis unit (Gibco BRL Life Technologies Inc.). The DNA was allowed to unwind for 20 min in electrophoresis running buffer solution (300 mM·NaOH, 1 mM·Na₂EDTA at pH >13; Sigma, St. Louis, MO). Electrophoresis was conducted for 20 min at 25 V and 300  mA (0.73 V/cm). All the technical steps were carried out under very dim indirect lighting. After electrophoresis, the slides were gently removed and rinsed with neutralization buffer (0.4 M·Tris pH 7.5, Sigma, St. Louis, MO); they were then dehydrated with ethanol (70%) and air-dried. All of the slides were coded before analysis. Ethidium bromide (75 μL of 20 μg/mL solution) was added to each slide, and a cover glass was placed on the gel. Individual nuclei were visualized under 400x magnification on a Leica DM4000 microscope.

Acetonitrile, ethyl acetate, formic acid and methanol were purchased from Kefo (Sisak, Croatia); Czapek Yeast Agar (CYA) was purchased from Oxoid (Hampshire, UK). Medium RPMI 1640, fetal bovine serum (FBS), phosphate buffered saline (PBS; without Ca²⁺ and Mg²⁺), trypsin-EDTA, antibiotics penicillin and streptomycin were from Lonza (Basel, Switzerland). The MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], acridine orange, ethidium bromide, normal melting point agarose (NMP), low melting point agarose (LMP), phorbol 12-myristate 13-acetate (PMA), Triton X-100 and Tris buffer were purchased from Sigma-Aldrich (Deisenhofen, Germany) and molecular biology grade dimethyl sulfoxide (DMSO), used for dissolving the extracts and for the control treatment, was purchased from Sigma-Aldrich (Deisenhofen, Germany); technical grade DMSO, used for dissolving the formazane, ethanol, sodium chloride, disodium-EDTA and sodium hydroxide were from Kemika (Zagreb, Croatia).

LC gradient-grade Methanol and formic acid were purchased from J. T. Baker (Deventer, The Netherlands). Methanol, carbon tetrachloride, and dimethyl sulfoxide were obtained from Sigma-Aldrich (Steinheim, Germany). Reference standards of the phenolic compounds were obtained from Sigma-Aldrich (Steinheim, Germany) and Extrasynthese (Genay, France). Hanks balanced salt solution (HBSS), low melting point agarose, normal melting point agarose, bovine serum albumin, phosphate-buffered saline (PBS), lysis buffer, sodium hydroxide (NaOH), ethylenediaminetetraacetic acid (EDTA), absolute alcohol, ethidium bromide and 1,1,3,3-tetraethoxypropane were purchased from Sigma-Aldrich (Steinheim, Germany).

Dulbecco’s modified Eagle's medium (DMEM), penicillin and streptomycin were obtained from Corning Cellgro (Manassas, VA, USA). Fetal bovine serum (FBS) was obtained from Gibco-BRL (Life Technologies, Grand Island, NY, USA). Amantadine HCl (A1260; Molecular Weight: 187.7), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PI, Triton X-100, ribonuclease A (RNase A), DTX, normal melting point agarose, low melting point agarose and fluorescein isothiocyanate (FITC)-dextran (40 kDa) were obtained from Sigma (St. Louis, MO, USA). Caspase-Glo 3/7 assay kit was purchased from Promega (Madison, WI, USA). Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit and Click-iT EdU Alexa Fluor 488 flow cytometry assay kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CFSE cell-division tracker kit was obtained from BioLegend (San Diego, CA, USA).