Ibitreat μ slide 6 0

ASF-2 cells were grown in DMEM (Lonza) with 10% fetal bovine serum (Thermo Scientific), 100 U/mL penicillin and streptomycin (Lonza) at 37°C, 5% CO₂ and 95% humidity. ASF-2 cells of passage 10 were detached from a tissue culture flask by trypsination with Trypsin EDTA (Lonza) and spun down at 600 g for 6 minutes. Cells were then resuspended in growth medium and 15.000 cells/well were seeded out in an ibiTreat μ-Slide VI 0.4 (Ibidi) and grown overnight. The next day the cells were rinsed with PBS, fixed in 4% PFA for 15 min., permeabilised with 0.025% Triton X100 (Sigma-Aldrich) for 10 min. and blocked with 2% BSA in PBS (BSA-PBS) for 1 hour at room temperature. The cells were incubated with 5 μg of purified LOB7 in 2% BSA-PBS or 100 μl of a 1:100 dilution of V9 antibody in 2% BSA-PBS (Sigma Aldrich) pr. well for 1 hour at room temperature. Visualisation of LOB7 was accomplished by incubation with a 1:100 dilution of Goat-anti-Rabbit Alexa Fluor 488 (Invitrogen, USA). V9 was visualised by a 1:100 dilution of Goat-anti-Mouse Alexa Fluor 546 (Invitrogen). Cell nuclei were stained with Vectashield Mounting Medium with DAPI (Vector Labs). Fluorescent images were obtained with a Leica DMI3000 B inverted microscope (Leica Microsystems).

Approximately 7000 MCF-7 cells were seeded into one channel of an ibiTreat μ-slide VI 0.4 (Ibidi, Gräfelfingen, Germany). The slide was temperature controlled and loaded into the FLUMIAS microscope (developed by FEI Munich GmbH [11 (link)]) shortly before the launch (Figure 12). Five minutes prior to launch three z-stacks were obtained from pre-selected cells as a ground control. About 75s after launch the microgravity phase was reached, and the microscope started recording the pre-selected cells. Three z-stacks were taken every one minute with 125 ms exposure time. The thickness of the z-stack was 21 μm with 0.5 μm step size. The procedure was repeated four times with a total number of five active phases covering 6 min of microgravity.
After recovery of the image data, a single image was extracted from each z-stack taken during microgravity to allow analysis of all images in the focal plane. The extracted images were deconvolved by Huygens Essential Scientific Volume Imaging software 4.3 and compared to a control image taken on ground.

A suspension of Ca9-22 cells (3 × 10⁵/ml) was seeded overnight onto ibiTreat μ-Slide VI ^0.4 (ibidi GmbH, Martinsried, Germany) and stimulated with 50 μg/ml of whole P. gingivalis cells at MOI of 0.13 for 4 d to assess intracellular IL-33 expression. The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with PBS containing 0.1% saponin for 10 min and then incubated in PBST containing 1% (w/v) BSA for 30 min to block non-specific binding. The cells were incubated for 1 h at 4°C with 0.5 μg/ml of phycoerythrin conjugated-rat anti-human IL-33 mAb (clone, 390412; R&D Systems, Minneapolis, MN). Thereafter, the cells were incubated with DAPI for 1 min to identify nuclei, mounted on slides using mounting medium, and assessed using fluorescence microscopy (BZ-9000, Keyence, Tokyo, Japan).