Coolpix 995 digital camera

Bone marrow cells were harvested from 4 male and 4 female littermate C57BL/6 mice at one month of age and seeded in α-MEM containing 15% heat-inactivated FBS at a density of 1×10⁶ cells/cm², and maintained in a humidified 5% CO₂ incubator at 37°C. To induce expression of Rankl by adherent osteoblastic cells and differentiation of mononuclear precursors into osteoclast-like cells, cultures were exposed for 7 days to 1,25 dihydroxyvitamin D₃, dissolved in 100% Et-OH and diluted to a concentration of 10 nM in α-MEM containing 15% heat-inactivated FBS [21] (link), [22] (link). Parallel control cultures were exposed to an equal volume of 100% Et-OH. To determine bone resorptive activity, cells were seeded on Osteo Assay Surface (Corning Life Sciences, Tewksbury, MA), cultured for 7 days under conditions favoring osteoclastogenesis and removed with bleach before von Kossa staining of the culture surface. Images of the stained surfaces were acquired with a Coolpix 995 digital camera (Nikon Inc., Melville, NY), imported in ImageJ v 1.48d (National Institutes of Health, Bethesda, MD) [23] and converted to 8-bit grayscale. Identical thresholds of gray intensity were set below the background of each image analyzed and resorbed area was defined as the area occupied by pixels with values of gray intensity below the preset threshold, determined with the measure function of ImageJ.

Sixty milliliters of ethanol (98–100%) were mixed with 40 mL of acetic acid (98–100%). Ten milligrams of Alcian blue 8 GX (stable solution for 1 year) were dissolved in this solution. One hundred and twenty milliliters of ethanol were mixed with 80 mL of acetic acid (98%–100%) to obtain the bleaching solution.
The process for the assay is shown as follows: the culture medium was gently aspirated from a multiwell taking extreme care not to aspirate the spheroid-like structure. It was washed twice with phosphate buffer saline (PBS) 1× without Ca⁺⁺/Mg⁺⁺. A sufficient amount of neutral buffered formalin (10%) was added to cover the spheroid-like structure, and the mixture was incubated at room temperature for 60 min. Formalin was carefully aspirated and washed 2 times in H₂O. Distilled water was carefully aspirated, and enough Alcian blue coloring solutions were added to generously cover the cartilage spheroids, as some evaporation occurred. The mixture was incubated overnight at room temperature and in the dark.
The Alcian blue stain solution was carefully aspirated, wash the cells were washed with the bleaching solution for 20 min. The washing step was repeated twice. The bleaching solution was carefully aspirated, and PBS was added.
The cells were then observed and photographed with a 5× magnification optical microscope equipped with a Nikon CoolPix 995 digital camera.

hTERT immortalised fibroblast cell lines were generated from chronic wound and patient matched normal fibroblast cell strains (strains described previously [22 (link)]). Fibroblasts were transfected with the hTERT containing retroviral vector pBABE-hTERT. Positively transfected cells were selected by the addition of puromycin to the growth medium (Fibroblast-Serum Containing Medium [F-SCM + Puro], consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with l-glutamine (2 mM), antibiotics (100 U/mL penicillin G; 100 µg/mL streptomycin sulphate; 0.25 µg/mL amphotericin B), puromycin 1 µg/mL, and 10% (v/v) foetal calf serum [FCS]; all of the reagents supplied by Invitrogen, Paisley, UK). Upon reaching 80–90% confluence, fibroblast populations were passaged at a ratio of 1:3.
The populations doublings (PDs) of the cell populations were derived by direct counting of cell numbers at each passage then calculation using the method previously described [54 (link)]. Cells in monolayer culture were digitally imaged 24 h after passage using a Nikon Coolpix 995 digital camera (Nikon, Kingston-upon-Thames, UK) attached to an Olympus CK2 microscope (×10 objective lens, F-Stop 3.5) (Olympus, Southall, UK).

¹H-nuclear magnetic resonance (NMR) spectroscopy using an MR400 DD2 (Agilent Technologies, Inc., Santa Clara, CA, USA) NMR spectrometer, differential scanning calorimetry (DSC) using a Q-10 (TA Instruments, Inc., New Castle, DE, USA), and polarized optical microscopy (POM) images of LC cells using a Nikon Eclipse E600 POL (NIKON, Inc., Tokyo, Japan) equipped with a polarizer and Nikon Coolpix 995 digital camera (NIKON, Inc., Tokyo, Japan) were employed for the characterization of the synthesized materials. The static contact angles of water on the polymer films were determined using a Kruss DSA10 (KRÜSS Scientific Instruments Inc., Hamburg, Germany) contact angle analyzer equipped with drop shape analysis software (KRÜSS Scientific Instruments Inc., Hamburg, Germany). The contact angles for each sample were measured more than four times on three independently prepared films, and the average values were used. The ultraviolet (UV) stability test of the LC cells was conducted using a VL-6.LC lamp (λ_max = 365 nm, Vilber Lourmat, Paris, France) with intensities of 5, 10, and 15 mW/cm² during the 30 min in order to corroborate the reliability to apply severe environment. The exposure dose of irradiated UV light on the LC cells was measured with a UV detector using GT-513 (Giltron, Seoul, Korea).