5 florouracil

The study involves use of analytical grade solvents i.e. n-hexane; chloroform; acetone; ethyl acetate; ethanol; methanol and dimethyl sulfoxide (DMSO) purchased from Sigma (Sigma-Aldrich, USA). The reagents include gallic acid; quercetin; 2,2-diphenyl-1-picryl-hydrazyl (DPPH); potassium acetate; aluminum chloride; Folin-Ciocalteu (F-C) reagent; sodium carbonate; ascorbic acid; ammonium molybdate; sodium phosphate; sulfuric acid; ferric cyanide; trichloroacetic acid and potassium ferricyanide procured from Merck (Merck KGaA, Germany), rutin; myricetin; caffiec acid;apigenin;kaempferol; catechin; and caffeic acid were purchased from Sigma (Sigma-Aldrich, Germany). Sabouraud dextrose agar (SDA), nutrient broth and nutrient agar media were purchased from Merck (Merck KGaA, Germany). RPMI-1640, fetal bovine serum (FBS) and medium 199 were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). Equipment included Agilent 1200 series binary gradient HPLC coupled with diode array detector (Agilent Technologies, Germany); CO₂ incubator (mco-17AIC,Sanyo-Japan); improved neubauer chamber (Marien, Germany). Reference standards employed in the current study, werepurchased from Sigma-Aldrich USA, include droxithromycin; cefixime; terbinafine; doxorubicin; surfactin; amphotericin-B; 5-Florouracil and vincristine.

To ensure the localization of Cy7MX in nuclei, 20,000 DLD1 cells were plated on glass coverslips in 6-well cell culture plates. DLD1 cells were incubated in 2 mL of media for 24 h. 2 mL of 1 mM 5-florouracil (F6627, Sigma–Aldrich) diluted in media or 2 mL of DMEM media were added to two groups of cells. At 96 h, media was removed, and the cells were washed with 1× PBS twice. Cells were then fixed with 2 mL of methanol at −20 °C for 10 min. Uracil DNA glycosylase (UDG) treatments in UDG reaction buffer—either 100 or 250 units—were added to each sample and incubated overnight. Cells were then washed with 1× PBS and incubated with a 25 µM Cy7MX probe for one hour while stirring. The samples were then rinsed with 1x PBS, stained with 4′,6-diamidino-2-phenylindole (DAPI), and soft mounted onto slides. Cells were imaged with a Leica STP 600 confocal microscope under 80x magnification. Excitation was performed at 405 nm and 633 nm with detectors measuring DAPI from 410–480 nm (32% gain) and Cy7MX from 650–680 nm (41% gain). Images were analyzed with ImageJ.