Eclipse ts2 fl inverted microscope

Cell were fixed in 4% paraformaldehyde or cold methanol, washed with PBS, permeabilized with PBS containing 0.2% Triton-X100 and blocked with PBS containing 0.2% Triton X-100 and 1% bovine serum albumin (BSA). Antibodies detecting HA tag (HA.11) were from Biolegend, anti-ORF50 was a gift from Carolina Arias (UC Santa Barbara, USA) and anti-EGFP was a kind gift from J. Mercer (Institute of Microbiology and Infection, University of Birmingham, UK). Secondary antibodies coupled to Alexa Fluor 488, 555 and 647 were from Thermo Fischer Scientific (Waltham, MA, USA). Nuclei were counterstained with Hoechst 33342 (1 µg/mL, Sigma, St. Louis, MO, USA). Images were acquired using an ImageXpress Pico microscope (Molecular Devices, San Jose, CA, USA), CellInsight High throughput microscope (Thermo Fischer Scientific) or Eclipse Ts2-FL inverted microscope (Nikon, Tokyo, Japan). Images were analyzed using the Cell Profiler 3.0 software package [27 (link)] and the following functions to construct the analysis pipelines: Identify Primary Objects, Relate Objects, Filter Objects, Measure Image Intensity, Measure Object Intensity and Export to Spreadsheet.

Cell migration and invasion were assessed using invasion assay. Briefly, cell culture inserts (24-well format, 8.0 μm pore size) were pre-coated with Matrigel (1:5 dilution; Corning). Transfected cells were then seeded at 10⁵ cells/insert in a serum free medium. The complete medium supplemented with 10% FBS was added to the lower chamber. All medium was applied with 10 μM sunitinib. After 48 h incubation, cell culture inserts were fixed with 100% methanol and stained with 0.3% crystal violet solution. The cells in the upper chamber were then removed with cotton swab and the inserts were mounted on glass slides. Microscopic images were acquired using an Eclipse Ts2-FL inverted microscope (Nikon) and the numbers of cells were counted using ImageJ software.

HUVEC were plated at a density of 1 × 104 cells per well in 96-well plates and treated for 72 h with heparin (5I U/mL), PRP (5–40%), PRP-HA (5–40%), PL (5–40%). Plates were washed three times with PBS (phosphate buffered saline), fixed with 4% paraformaldehyde for 10 min, stained with 0.1% crystal violet, and samples were rinsed in running water and air-dried. Images were taken under an Eclipse Ts2-FL inverted microscope fitted with a DS-Fi3 camera and DS-L4 control unit (Nikon corporation, Tokyo, Japan). Samples were dissolved with glacial acetic acid, and the optical density at 570 nm was determined using an automatic microplate reader (Biotek).