N tetraacetic acid

All reagents were of analytical grade or of the highest grade available. Antibiotic mixture of penicillin/streptomycin (10,000 U/mL/10,000 mg/mL), fungizone (250 mg/mL), and heat-inactivated fetal bovine serum (FBS) were obtained from GIBCO Invitrogen (Barcelona, Spain). Collagen G was obtained from Merck (Darmstadt, Germany). 4-Fluorobenzaldehyde (≥98%), collagenase from Clostridium histolyticum Type IA, desmosterol (≥84%), dexamethasone, ethylene glycol-bis-(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA), gentamicin, insulin solution from bovine pancreas (10 mg/mL), methoxyamine hydrochloride (≥98%), N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + 1% TMCS), O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA, ≥99%), sodium chloride (NaCl, ≥99.5%), thiazolyl blue tetrazolium bromide (MTT, ≥98%), thymol (≥98.5%), Triton X-100, trypan blue solution, Williams’ E medium, and all standards used throughout the work were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Methanol (≥99.9%) and pyridine (≥99%) were purchased from VWR (Leuven, Belgium).

The comet assay was performed using a Trevigen Comet Assay Kit (Trevigen Inc., Gaithersburg, MD, USA) to detect DNA strand breaks at the single-cell level as described previously [18 (link)]. Briefly, cells were exposed to millimeter-wavelength radiation for 24 h, collected by trypsinization and centrifuged immediately after exposure, then mixed with low melting point agarose to prepare a cell suspension in 0.1% agarose/phosphate buffered saline (PBS). After gelation of the agarose, the cells were lysed, then the alkaline unwinding was performed (1 h at 4 °C, pH > 13). The resulting DNA samples were electrophoresed at 1 V/cm for 30 min in a 0.3 M NaOH (Nacalai Tesque, Kyoto, Japan) and 1 mM ethylenediamine-N,N,N’,N’-tetraacetic acid (Sigma-Aldrich) solution. After the DNA was stained with SYBR Green I, immunofluorescence images were captured using a fluorescence microscope (Olympus, Tokyo, Japan). DNA strand breaks were analyzed using Comet software (Perceptive Instruments, Suffolk, UK). At least 100 comets from each gel were analyzed, and at least five independent experiments were performed. Tail length indicates the pixel length of the comet tail, tail percent indicates the percentage of tail content relative to comet content, and tail moment was calculated as follows: