Axio observer inverted fluorescence microscope

Cells in the monolayers were fixed in a buffered formalin solution. Non-specific binding was blocked with a 3% (w/v) BSA solution and cells were then incubated overnight at 4°C with primary antibodies rabbit anti-human laminin (1:30) (Abcam, United Kingdom), rabbit anti-human fibronectin (1:50) (Abcam, United Kingdom) and rabbit anti-human type I collagen (1:50) (Abcam, United Kingdom). After repeated washes in PBS, secondary antibody Alexa Fluor 488 donkey anti-rabbit (1:500) (Molecular probes, USA) was incubated with cells for 45 minutes at room temperature. Cells were washed in PBS and cell nuclei was counterstained with DAPI. Cell monolayers were then analyzed in a Zeiss Axio Observer inverted fluorescence microscope (Zeiss, Germany) and images acquired using ZEN 2 software (Zeiss, Germany).

Changes to drug target binding were quantified using a competitive binding assay between Gem-Atto and the parent drug, gemcitabine, on the established human pancreatic cancer cell lines: PANC-1, AsPC-1 and Capan-1. The cells were washed, trypsinized and plated in a 96-well glass bottom plate (Cellvis, Mountain View, CA) at a concentration of 0.1x10⁶ cells/ml. The cells equilibrated for 24 hours after which, gemcitabine-HCl (Sigma-Aldrich) was added to all cell lines at 0, 0.005, 0.05, 0.5, 5, 50 μM in triplicate wells. Every well also contained Gem-Atto at a single concentration (500 nM). Following a 24-hour incubation, the cells were gently washed 3 x 5 min with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) for 15 min, washed again with PBS for 5 min and imaged in fresh PBS on a Zeiss AxioObserver inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany). For fluorescence excitation, a PhotoFluor II broad band light source (89 North, Burlington, VT) was filtered using a 650 ± 22.5 nm bandpass excitation filter and a 720 ± 30 nm bandpass emission filter to acquire images for Atto680. Individual cellular fluorescence was quantified using FIJI and analyzed with Prism (GraphPad, San Diego, CA).

Immunofluorescence was used to assess the in-situ expression of the FRY. MDA-MB-231 related cells were cultured on coverslips in DMEM medium with 10% FBS and penicillin/streptomycin at 37°C with 5% CO₂ for 24 h. The cells were briefly washed in PBS, fixed with 4% formaldehyde for 30 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min and incubated in blocking buffer (10% goat serum, 0.2% Triton X-100, 0.05% Tween-20 in PBS, pH 7.4) for 1 h at room temperature. The cells were then incubated with primary antibodies diluted in blocking buffer overnight at 4°C. After rinsing, the primary antibodies were detected with Alexa Fluor® 594 conjugated goat anti-mouse IgG (Cell Signaling Technology) or Alexa Fluor® 555 conjugated goat anti-rabbit IgG (Cell Signaling Technology) secondary antibodies diluted at 1:500 in blocking buffer for 1 h at room temperature. Coverslips were mounted using ProLong® Diamond Antifade Mountant with DAPI (Life Technologies) and fluorescence images were visualized and captured with a ZEISS Axio Observer inverted fluorescence microscope (ZEISS, Jena, Germany).
Primary antibodies and dilution used in applications include the following: mouse anti-FRY (2F2-D8-E3-G3) antibody (RayBiotech, 1:100), mouse monoclonal anti-β-Actin antibody (Sigma-Aldrich, 1:2,000).