Powershot s50 camera

Specimens are preserved in 70% alcohol, deposited at the Bombay Natural History Society (BNHS), Mumbai. Specimens were studied under a Leica stereozoom (MZ6) microscope, photographed using mounted Canon Powershot S50 camera, assembled using Combine ZM software and the images were processed with Adobe Photoshop CS5. Measurements were done with Erma stage and ocular micrometer and an accurate scale. Epigyna were cleared in 10% KOH and kept in Polyvinyl Lactophenol (PVLP) gel with Lignin pink stain for seven days before imaging. All measurements are in millimetres; measurements of other specimen of P.octomaculatus are provided in parentheses. Map was produced with DIVA-GIS v. 7.5c, with geographical coordinates obtained from Google Earth.

Spheroids were generated following the protocol described by Naakka et al. [41] (link). Briefly, the cells were seeded in 50 µl complete culture medium (see Cells and cell culture) at a cell density of 1 × 10³ cells/well into ultra-low attachment 96-well round bottom plates (Corning) and incubated at 37 °C for 4 days. After visual confirmation, spheroids were embedded in 50 µl gel containing 0.5 mg/ml Myogel [19] (link) or OmGel, 0.5 mg/ml Fibrinogen (Merck), 0.3 U/ml Thrombin (Sigma-Aldrich) and 33.3 µg/ml of Aprotinin (Sigma-Aldrich) in complete culture medium of each cell line. For Matrigel (BD Biosciences) wells, the gel was diluted to 0.5 mg/ml with the complete culture medium of each cell line. The plate was then transferred to the incubator at 37 °C for 30 min to allow the gel to solidify. 100 µl of complete culture medium was added on top of the gels. Spheroids were imaged at 0 h and after 4 and 9 days of incubation at 37 °C, using a Nikon Eclipse TS100 inverted light microscope, with 4x objective magnification, connected to a Canon PowerShot S50 camera. Fiji software [42] (link) was used for measuring the area covered by spheroids. The fold change in the total spheroid area at each time point compared with the area at 0 h was calculated.

CGF-released cells were suspended in M199 medium complete, containing 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin and seeded in appropriate cell plate coated with 1% gelatin. In our experimental conditions, CGF-derived cells adhered to the culture plate and grew until they reached the confluence. CGF-derived cells were phenotypically characterized by hematoxylin–eosin staining and immunostaining analysis (Figure 1). CGF cells, grown in 24-well plates on coverslips (Thermanox, ProSciTech, Thuringowa, Queensland, Australia), were fixed with cold methanol for 10 min on ice and washed with PBS; then, hematoxylin–eosin staining and immunostaining analysis were performed. For immunocytochemistry, monolayers were incubated overnight with antibodies against endothelial nitric oxide synthase (eNOS), VE-cadherin, and CD34 at concentrations of 1 μg/mL and with the antibody against VEGFR-2 at a concentration of 0.2 μg/mL. After 3 washes with PBS, monolayers were incubated for 1 h with a biotinylated anti-mouse (for eNOS, VE-cadherin) or anti-rabbit (for VEGFR-2, CD34) IgG antibody (Santa Cruz), and for 1 h with extravidin peroxidase (Merck). Monolayers were then incubated with diamminobenzidine (Merck) for 30 min, washed, and directly mounted, and pictures (×100 magnification) were taken with a digital output Canon Powershot S50 camera.

The DNA damage was assessed using the SCGE method, also known as comet assay, as previously described [22 (link)]. The fibroblast cells were briefly harvested from suspension and then embedded in a thin layer of agarose gel (1% low melting) upon clear microscope slides. Cells were lysed and DNA was unwound under alkaline conditions (1 mM EDTA and 300 mM NaOH–pH 13) for 20 min. Afterwards, electrophoresis was employed for 30 min at 300 mA and 25 V (*0.86 V/cm). Following electrophoresis, the slides were washed with 0.4 M tris(hydroxymethyl)aminomethane (Tris) (pH 7.5) and stained with ethidium bromide (2 µg/mL). Analysis was carried out by using a DM IRB fluorescence microscope (Leica Microsystem, Heidelberg, Mannheim, Germany), equipped with a digital recording Power Shot S50 camera (Canon, Milan, Italy). Two replicas for each batch were carried out. Images of 50 cells per slide (100 cells per batch) were randomly acquired and analyzed using CASP software (v.1.2.3, Casplab, Wrocław, Poland). The featured parameters were: length (TL), percentage of DNA (% T-DNA) and moment (TM) of the tail. All values were statically evaluated by one-way ANOVA (Analysis of Variance).