Fluorescein diacetate

Differential staining with specific fluorochromes was used to distinguish living and dead cells. Cells were trypsinized and suspended in 100 μl PBS and 100 μl of a solution containing 25% PI (1 mg ml^-1 in water), 50% fluorescein diacetate (1.5 mg ml^-1 in DMSO), 10% HO (1 mg ml^-1 in water) (Sigma-Aldrich Co. LLC, St. Louis, USA) and 15% PBS. Ten microliters of the samples were placed in glass slabs and covered with coverslip. The area of analysis was 0.030 mm². Cells were classified as either viable (spherical blue nucleus stained by HO, green cytoplasm stained by fluorescein diacetate, excited at 360 nm), non-viable cells (blue nucleus with apoptotic bodies stained by HO, green cytoplasm) or necrotic (red enlarged nucleus with spherical vesicles stained by PI, excited at 538 nm) using confocal microscopy (63x Leica TCS_SPE). The non-viable and necrotic cells were considered to be dead. The percentages of living and dead cells were quantified [20 (link),21 (link)].

Gels were immersed in PBS containing 20 μg of fluorescein diacetate (Sigma-Aldrich) and 4 μM ethidium homodimer (Sigma-Aldrich) for 10 min at 37°C, after which they were placed on a glass slide. Gels were imaged using a Zeiss LSM610 confocal microscope.

To determine the kinetics of our compounds activity against E. histolytica, we adapted the viability assay we developed in our previous studies [6 (link)] and performed a time course to measure changes in the number of live trophozoites over 48 hours. E. histolytica trophozoites (10,000 parasites in 150μl media) were seeded into 96-well plates and allowed to grow overnight in an anaerobic chamber. The next day, a single plate was removed for analysis, and drug was added to remaining plates at 2x the previously calculated EC₅₀ concentration. Parasite viability was assayed at 10, 24 and 48h using the live cell marker fluorescein diacetate (Sigma) as previously described [6 (link)]. Three independent biological replicates were performed.

Tenocytes seeded on Poly-L-lysin (Biochrom)-coated cover slides and stimulated with the agents for 72 h were incubated in a mixture of 5 µL/mL fluorescein diacetate (Sigma-Aldrich, 3 mg/mL dissolved in acetone (stock solution)) and 1 µg/mL ethidium bromide (Carl-Roth, Karlsruhe, Germany) diluted in 1 mL phosphate-buffered saline (PBS) for 10 min. The green (vital) or red (dead) cell fluorescence was visualized using fluorescence microscopy (Axioskop 40, Carl Zeiss, Jena, Germany) using a digital camera (Color View II, Olympus, Shinjuku, Japan). Three microscopic fields of each treatment group in each independent experiment were evaluated using ImageJ for vital and dead cells.

Rat tail type I collagen, Transwell inserts (0.9 cm² cell culture area, 1.6 × 10⁶ pores/cm²), 12-well polystyrene tissue culture dishes, 12-well polycarbonate plates and Transwell inserts (1.12 cm² cell culture area, 1.6 × 10⁸ pores/cm²) were purchased from Corning (Corning, NY). SB202190 was from Selleckchem (Houston, TX). Collagenase (type IV) was purchased from Worthington Biochemicals (Lakewood, NJ). Y-27632 and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from ApexBio Technology (Houston, TX). Human collagen (type I) was obtained from Advanced Biomatrix (Carlsbad, CA). The ALP assay kit was from AbCam (Cambridge, MA). Gastrin was from Anaspec (Freemont, CA). UGT-Glo™ Assay and P450-Glo™ CYP3A4 Assay were obtained from Promega (Madison, WI). N-acetyl cysteine was purchased from MP Biomedicals (Santa Ana, CA). Murine EGF was procured from Peprotech (Rock Hill, NJ). Primocin was purchased from InvivoGen (San Diego, CA). Nicotinamide, L-alanine p-nitroanilide, fluorescein diacetate, loperamide, zafirlukast, cobalt chloride, potassium phosphate buffer, bovine serum albumin, and A83–01 were acquired from Sigma-Aldrich (St. Louis, MO). All other reagents and Bicinchoninic Acid (BCA) Protein Assay Kit were purchased from Thermo Fisher Scientific (Waltham, MA).

Dead/live staining was used to analyse cell viability after culturing in the scaffolds for 3 days. Briefly, cell-scaffold complexes were stained with FDA (fluorescein diacetate, 5 μg/ml, Sigma) and PI (propidium iodide, 5 μg/ml, Sigma) for 5 min and rinsed 3 times with PBS (phosphate-buffered solution, pH 7.4). The samples were immediately visualized using a laser scanning confocal microscope (Olympus FV 1200, Japan) [26 (link)]. Red and green fluorescence indicate dead and live cells, respectively.
To observe the distribution of seed cells in the 3D-oriented scaffold, scanning electron microscopy (SEM) was applied to view the samples in the sagittal plane and in cross sections after culturing for 3 weeks. All samples were fixed in 2.5% glutaraldehyde, dehydrated in a series of graded ethanol to 100% ethanol, treated with hexamethyldisilazane and sputter-coated with gold-palladium before observation [21 (link)].