Imagexpress micro confocal high content microscope

Immunofluorescence was performed after fixing cells grown on glass coverslips, or directly in 96-well dish plates for high-throughput microscopy, for 20 min with 3% PFA in PBS. All steps of the immunofluorescence procedure were performed at room temperature. When indicated, cells were permeabilized for 5 min with 0.01% saponin in PBS before PFA fixation. After fixation, cells were incubated for 45 min in 1% fish skin gelatin and 0.1% saponin in PBS, followed by 30-min incubation with the primary antibody in 1% fish gelatin in PBS. After washing the primary antibody with PBS, the cells were incubated for 30 min with the secondary antibody (Cy2-, Cy3-, or Cy5-conjugated fluorescent antibodies) in 1% fish gelatin in PBS, followed by PBS washes. The cells were mounted in Mowiol 40-88 medium containing 10 µg/ml Hoechst and imaged with a Zeiss 700 confocal microscope (Carl Zeiss) using a 63× 1.4-NA oil differential interference contrast (DIC) Plan-Apochromat objective (Nikon) and Zen imaging software (Carl Zeiss). For high-throughput microscopy, 96-well plates were imaged with an ImageXpress Micro XLS Widefield High-content microscope (Molecular Devices) or an ImageXpress Micro Confocal High-content microscope (Molecular Devices; used in the wide-field mode) using a 40× 0.95-NA objective (Nikon).

5637 cells treated with siRNAs were seeded at a density of 50000 cells/well in 96-well glass bottom plates (Greiner Sensoplate, M4187-16EA, Merck) and placed in a CO₂ incubator overnight. Prior to imaging, cells were stained with NucBlue solution (Invitrogen) to label the nuclei of live cells. In brief, cells were incubated at 37°C and 5% CO₂ for 30 min with NucBlue solution diluted 1:40 in culture medium. Subsequently, cells were washed twice with pre-warmed culture medium. Image acquisition was carried out in an ImageXpress Micro Confocal high-content microscope (Molecular Devices) in widefield mode. Time lapse series were acquired for a total time period of 4.5 h using a time interval of 4 min between frames, a 20x 0.45 NA Ph1 air objective, filter set for detection of DAPI and phase contrast, and an environmental control gasket that maintain 37°C and 5% CO₂. Cell displacement was analyzed by particle tracking using the TrackMate plugin in Fiji ImageJ (20 (link)) in combination with in-house Python-based script (Python 3.7.6). Average displacement speed was calculated as the average of all tracked cell displacements within a 4 h period of imaging.

Urinary bladder cancer cell lines (SV-HUC-1, 5637, SW780, T24, 5HT1376, HT1197) were seeded at sub-confluent densities (4000 cells/well) in 96-well glass bottom plates (Greiner Sensoplate, M4187-16EA, Merck). After seeding, the plates were placed in a CO₂ incubator overnight to allow cells to attach to the glass surface. Subsequently, cells were monitored using an ImageXpress Micro Confocal high-content microscope (Molecular Devices), equipped with an environmental chamber maintaining 5% CO₂ and 37°C, and controlled by the MetaXpress 6 software. Time lapse series of phase contrast images were acquired in widefield mode using a 20x 0.45 NA Ph1 air objective, camera binning = 2, a time interval of 3 min between frames and a total imaging period of 20 h. Cell displacement were analyzed by particle tracking using the TrackMate plugin in Fiji ImageJ (20 (link)) in combination with in house Python-based script (Python 3.7.6). Average displacement speed was calculated as the average of all tracked cell displacements within a 12 h period of imaging.

Imaging of embryos was performed 24 hpi and 72 hpi at 32–34 °C using the ImageXpress Micro Confocal High-Content Microscope (Molecular Devices, San José, CA, USA). Embryos were anesthetized using 1x tricaine and were put into Hashimoto (Funakoshi Co., Ltd., Tokyo, Japan) zebrafish 96-well plates with one embryo/well. Images were taken with 4x magnification in multiple z-stacks (35 z-stacks per image; step size 20 µM) and two sites per well covering the whole yolk sac, using brightfield and Cy3 settings, respectively. The tumor volume was calculated with an automated analysis pipeline (in-house macro for ImageJ), carefully discriminating specific signals from yolk sac-caused blurry background signal, as described previously [26 (link)]. To determine the difference (e.g., increase due to growth) of tumor volume from baseline until end of treatment, the percentage of volume difference was calculated between these two imaging time points.
The zebrafish-adapted Response Evaluation Criteria in Solid Tumors (RECIST) as described previously [26 (link)] was used to evaluate and visualize the drug response. To be classified as progressive disease (PD) the tumor volume must have increased at least 20%, and to be classified as partial response (PR) the tumor volume must have decreased by more than 30%.