Cq1 spinning disc confocal microscope

Sub‐confluent monolayers of MEFs cultured on optical 96‐well plates (Greiner 655090) in DMEM 10% FBS were incubated with IAV X31 on ice for 1 h in infection medium (DMEM, 50 mM HEPES pH 6.8, 0.2% BSA) and warmed for 2 min at 37°C by addition of warm medium (pH 6.8) or warm medium adjusted to low pH (pH 5.0) by citrate buffer, on a metal block immersed in a 37°C water bath. Another warm metal block was placed on top of the plate. The medium was replaced with STOP medium (DMEM, 50mM HEPES pH7.4, 20 mM NH₄Cl) to block viral entry through the endocytic route, and the plate was transferred to 37°C and incubated for 18 h, fixed with 4% paraformaldehyde in PBS. Fixed cells were blocked for 1 h with 1% BSA in PBS before incubation with a mouse anti‐nucleoprotein monoclonal antibody (HB‐65, ATCC) for 1h at RT, followed by a 30‐min incubation with a second goat anti‐mouse‐Alexa Fluor 647 antibody (Invitrogen, A21235), together with Hoechst 33342 (Thermo Fisher Scientific). Image acquisition was performed with a Yokogawa CQ1 spinning disc confocal microscope, using a 10x air objective with an NA of 0.40, in 2‐channel mode. Image analysis was performed using the Cell Path Finder version 3.04.03.02 (Yokogawa Electric Corporation).

X31/R18 double‐labelled X31 was prepared as herein described. For the fusion experiment, MEF cells were incubated in binding media for 1h on ice and then warmed for 1h at 37°C by addition of warm binding media and transfer of the plates to 37°C, followed by fixation with 4% paraformaldehyde in PBS. The cells were then labelled with Hoechst and WGA‐AF647 as herein described. Image acquisition was performed with a Yokogawa CQ1 spinning disc confocal microscope, using a 40x air objective with an NA of 0.95, in 4‐channel mode. Image analysis was performed using the Cell Path Finder version 3.04.03.02 (Yokogawa Electric Corporation).

Image acquisition was performed with a Yokogawa CQ1 spinning disc confocal microscope, using a 10x air objective with an NA of 0.40, or a 40x air objective with an NA of 0.95. Imaging was performed with up to four excitation laser lines (405/488/561/640nms) with spinning disc confocal. For 10x objective, images were acquired with 5 z‐stacks to cover 40 µm; for 40x objective, images were acquired with 20 z‐stacks to cover 30 µm. Maximum intensity projected images were used for image analysis using the Cell Path Finder version 3.04.03.02 (Yokogawa Electric Corporation).