Csu x1 spinning disk confocal microscope

Dispersal of GBM cells through a NHA-seeded porous filter was measured as previously described [1 (link)]. Two-hundred μm thick, cross-linked polystyrene scaffolds (Alvetex, Reinnervate, Durham, UK) with tunnel diameters of 8–13 μm were seeded with 1x10⁶ NHA cells in 100 μL of tissue culture medium. After 60 min to allow NHA cells to adhere, scaffolds were placed in 12-well plates and incubated in 4mls of TCM for 48 h to permit incorporation of NHA cells throughout the scaffold. After 48 h, GBM cells that had been transfected with BacMam 2.0 GFPT (Life Technologies, Long Island, NY) were deposited onto each scaffold in a small volume of medium. Scaffolds were incubated for 48 h to allow time for tumor cells to infiltrate and disperse. To image dispersed cells, a Yokogawa CSU-X1 spinning disk confocal microscope with MetaMorph software was used to generate z-stacks of images taken at 1 μm intervals. Differential interference contrast microscopy was used to identify the z = 0 starting point for each z-stack. The z-axis position of each cell within each tissue-scaffold was scored. Within any given scaffold the mean average z-axis cell position from 5– 6 z-stacks was measured and recorded.

For confirmation of protein localization, strains were grown in synthetic complete media (minus prototrophic nutrients) with 2% glucose overnight, back diluted 50-fold into fresh media, and grown for an additional 6 h. Cultures were concentrated by centrifugation, washed in pH 7.4 PBS and spotted onto plain glass slides to be examined with a confocal fluorescence microscope.
Strains 9, 10 and 40–51 were examined with confocal microscopy using a CSU-X1 spinning disk confocal microscope (Yokogawa) with Leica optics including a × 100 objective and 442, 488 and 561 nm excitation lasers for cyan fluorescent protein (CFP, specifically mTurquoise2), YFP (Venus) and RFP (mKate2), respectively. Images were taken using a QuantEM 512SC EMCCD camera (Photometrics). All images were analysed using Fiji (http://fiji.sc).

mAID-Sp1; H2B-mCherry cells were grown in an 8 well Falcon Chambered Cull Culture Slides (Corning 354108) for at least 24 h. Two hours prior to imaging, cells were treated with 500 µm auxin. Images were acquired at 20 × magnification every 3 min using the Evos FL auto microscope with the on-stage incubator maintaining 37 °C. hTERT RPE-1^{TdTomato−CENP−A} cells were grown in an 8 well µ-Slide 8 tissue culture plate (ibidi 80806) for 24 h and transfected with Sp1-GFP plasmid using Lipofectamine LTX with Plus reagent (ThermoFisher A12621) and allowed to recover for 48 h. One drop of NucBlue Live ReadyProbe (ThermoFisher R37605) was added directly to the media to label DNA in live cells, which were subsequently imaged using the Nikon Ti Eclipse, Yokogawa CSU-X1 Spinning Disk Confocal microscope at 60 × magnification with an on-stage incubator maintaining 37 °C.