Dfc9000 gt camera

To image embryos and adults, L4-stage animals expressing either nIs470, nIs500, or burIs2 were placed onto NGM agar plates seeded with either E. coli HB101 or P. vranovensis BIGb446 at room temperature (22 °C) for 24 h. Embryos and adults in Fig. 4c and Supplementary Fig. 4 were collected and immediately imaged using a Zeiss AXIO imager A1 microscope and a Hamamatsu ORCA-ER camera. Embryos and adults in Supplementary Figs. 3 and 4 were imaged using a Leica DM6 B and a Leica DFC9000 GT camera. To image L2-stage larva in Supplementary Fig. 4, embryos collected from parents exposed to P. vranovensis were allowed to develop on plates seeded with E. coli HB101 for an additional 24 h at room temperature (22 °C) and then imaged using a Leica DM6 B and a Leica DFC9000 GT camera.

Images were obtained using a Leica DMi8 microscope with a PE4000 LED light source, DFC9000GT camera, and LAS X imaging software. A549 cells were seeded in 3 cm tissue culture plates at a density of 50,000 cells per well in DMEM containing fluorescent organic salts at indicated concentrations. The cells were incubated for 2 days at 37 °C with 5% CO₂ until the day of imaging. For live cell imaging, the media was aspirated, and the cells were washed with phosphate buffered saline (PBS, Sigma-Aldrich) 5 times before being imaged in PBS.
For colocalization analysis, A549 cells were grown on 0.5 mm coverslips placed in 3 cm tissue culture plates containing media for 3 days. Cells were then fixed by aspirating media, washing with PBS 5 times, then submerging the coverslip in cold methanol and incubating on ice for 15 minutes. The fixed cells were stained with 1 µM 2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]−2,5′-bi-1H-benzimidazole trihydrochloride trihydrate (Hoechst 33342, Invitrogen) for 5 minutes, washed with PBS, and then incubated with 15 µM of 3,6-diamino-9-(2-(methoxycarbonyl)phenyl chloride (Rhodamine123) and 1 µM CyPF₆ for 15 minutes before being washed and mounted to slides with Fluoromount-G (Invitrogen). Cells were analyzed using a Leica DMi8 microscope with a PE4000 LED light source, DFC9000GT camera, and LAS X imaging software.

To induce and observe cell migration, 3D-µ-Slide migration-chambers (Ibidi GmbH, Graefelfing, Germany) were used. One slide contains three separated systems or chambers, each chamber consists of a central channel and a left and a right reservoir (Figure 3).
The three chambers were filled as described in Table 1 and Figure 3b. In this setup, the PMNs—placed in the right reservoir—migrated along the gradient of the chemoattractant n-formylmethionyl-leucyl-phenylalanine (fMLP; Sigma Aldrich, St. Louis, USA), which was brought into the left reservoir. During migration, cells had to pass through the collagen matrix inside the central channel and could thereby be examined via live cell imaging (Figure 3c). Based on preliminary tests (data not shown), where we had already proven that only granulocytes were able to pass the collagen matrix, we were assured that solely this cell type (CD11b⁺; CD62L⁺; CD66⁺) is observed.
Cell observation was performed with a Leica DMi8 microscope and recorded with Leica DFC9000 GT camera (both Leica Microsystems GmbH, Wetzlar, Germany) for 22 h. Both camera and microscope were controlled by Leica Application Suite X software platform, version 3.4.2.18368 (Leica Microsystems GmbH). A stage top incubator consisting of Ibidi Blue Line gas mixer and Ibidi heating chamber (both Ibidi GmbH) was used to keep conditions constant at 37 °C and 5% CO₂.