Mn 151 micromanipulator

BHK cells were grown to a confluent adherent monolayer, washed with PBS, detached with trypsin, resuspended in DMEM supplemented with 2% FBS, inoculated with VSV at the indicated MOI, and incubated for 45 min at 37°C to allow for viral adsorption. The inoculated cells were then diluted 1/20 in the same medium and placed in a 60-mm dish to isolate individual cells with a G-1 glass capillary (Narishige) made to fit the diameter of BHK cells and set onto a Narishige MN-151 micromanipulator under an inverted microscope. Individual cells were focused at 100 × magnification, aspirated in the glass capillary, and immediately re-injected in a drop of 10 μl DMEM using an O₂-supplied Narishige IM-300 micro-injector. Drops containing single cells were pipetted and released into 100 μl of infection media in an assigned well of a 96-well plate, and cells were incubated 24 hr to allow for viral replication and progeny release. Aliquots of the supernatant were then stored at −70°C. The single-cell isolation procedure was repeated to ascertain the possibility of carry-over contamination from viruses present in the inoculum. The number of PFUs per well was clearly bimodal, with either one or zero PFU/well or > 100 PFU/well, thus indicating that < 1% of the plaques could originate from the viral stock used for inoculation (Figure S4).

To test the effects of CRPs and IL-6 in the process of autophagy in vivo, groups of 30 one-cell-stage embryos of GFP-LC3 zebrafish^{101 (link)} were microinjected with 2 nL of PBS containing 150 pg of either pMCV1.4, pMCV1.4-crp1, 4 or 5 or pMCV1.4-il6. The microinjections were performed with glass microcapillary pipettes (WPI, Sarasota, FL, USA) incorporated into an MN-151 micromanipulator and an IM-30 microinjector (Narishige, Tokyo, Japan). The treated 3-day-old hatched larvae were anaesthetized (by adding 200 µL of 0.05% MS-222 solution to a Petri plate with 10 mL of water) and photographed using a Multi‐Zoom AZ100 microscope equipped with a DS-Ri1 digital camera (Nikon, Melville, NY); the images were processed with LAS AF software (Leica).