Sp5 inverted

Cells, seeded onto gridded dishes (MatTek Corporation, P35G-2-14-C-GRID), were transfected with GFP-NP and simultaneously infected or mock-infected with PR8 at an MOI of 10. At indicated times, cells were fixed, imaged at the confocal microscope Leica SP5 Inverted and finally processed for electron microscopy imaging, as described previously^{14 (link)}. Sections of 70 nm thickness were cut using a Leica EM-FC7 Ultramicrotome. The regions of interest were acquired with a Hitachi H-7650 operating at 100 keV equipped with a XR41M mid mount AMT digital camera. Images were post-processed using Adobe Photoshop CS5 and ImageJ (NIH).

All images were acquired on a Leica SP5 inverted or Leica SP8 upright confocal microscope, using either a 40 × 1.3 NA PL Apo or 40×/1.3 HC PL Apo CS2 Oil objective, respectively. All wing discs were imaged as z-stacks with each section corresponding to 1 μm. Clone areas were measured on a medial section of the pouch region either manually or using a custom script in Fiji^{75 (link)}. For cell death quantifications, cells that were positive for cleaved caspase-3 (by immunostaining) and within the pouch region were counted. The total number of dying cells was normalised to the respective clone area/perimeter. Caspase-positive cells within a 2-cell diameter of the periphery of a clone were classed as ‘clone border’, whereas those further within were classed as ‘clone centre’. Both counts were then normalised to their respective areas. Images were analysed and processed using Fiji (version 2) and Photoshop (Adobe version CS6).