Te200 inverted epifluorescence microscope

HUVEC cells were utilized for a tube formation assay as per the protocol of the manufacturer in a µ-slide assay format. Briefly, IBIDI microplates (81506 Ibidi, Germany) were loaded with 10µL of Matrigel™ (354230, Corning, USA) and incubated for 1hr to allow for Matrigel™ polymerization. HUVEC cells were plated in triplicates at 1x10⁴ cells/well above the polymerized Matrigel™ in 50µL of media with one of the following treatments: VEGF, PBS, LIF (1, 20, 100ng/mL). Cells were incubated at 37°C and with 5% CO₂ for 4hrs. Images were taken on a Nikon TE200 inverted epifluorescence microscope using a 10× objective and a cooled CCD camera and analyzed by WimTube: Tube Formation Assay Image Analysis Solution (33 ).

Vero, HaCaT, L/EGFP, HeLa and HeLa/EGFP-UL31 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and grown at 37 °C in a 5% CO₂ environment. HeLa/EGFP-UL31 were constructed by transfecting HeLa cells with linearized plasmid encoding EGFP-UL31 and selecting for G418 resistant cells in medium containing 400 μg/ml G418. G418 resistant cells stably expressing EGFP-UL31 were identified and isolated with the aid of a Nikon TE200 inverted epifluorescence microscope and were maintained in medium supplemented with G418. HSV-2 strain HG52 was propagated on Vero or HaCaT cells.

To analyze macroscopic plaque formation, monolayers of Vero cells were infected with equivalent numbers of plaque forming units, overlaid with semisolid medium containing 1% methyl cellulose, then fixed and stained with 70% methanol containing 0.5% methylene blue at 72 hpi. To analyze cell-to-cell spread quantitatively, monolayers of Vero cells growing on glass bottom dishes (MatTek, Ashland, MA, USA) were infected with equivalent numbers of plaque forming units. At 24 hpi, infected monolayers were fixed with 4% paraformaldehyde and stained for the presence of the HSV kinase Us3, as described previously [16 (link)]. Images of plaques were captured on an Olympus FV1000 laser scanning confocal microscope using a 10× objective or on a Nikon TE200 inverted epifluorescence microscope using a 10× objective and a cooled CCD camera. To quantify these results, the numbers of pixels in the area of each plaque were counted using Image-Pro 6.3 software (Media Cybernetics, Bethesda, MD, USA). Results shown were derived from 40 distinct plaques per strain. Single infected cells were not included in this quantification.