Opera qehs high content screening platform

A PerkinElmer (Waltham, MA) Opera QEHS High-Content Screening platform was used for fully automated confocal collection of images. This system employed a 40x water immersion objective lens, laser illuminated Nipkow disk, and cooled CCD cameras to digitally capture high-resolution confocal fluorescence micrographs (300 nm pixel size with 2×2 camera pixel binning). An image analysis pipeline was customized using the Columbus software (PerkinElmer) to automatically segment the nucleus using the DRAQ5 channel and then construct a ring region (cytoplasm) around the nucleus mask for each cell in the digital micrographs. The pipeline filtered only for cells expressing GFP-GR- TRβ out the cells then measured the mean GFP-GR- TRβ intensity in both compartments in the GFP channel, and translocation was calculated as a ratio of these intensities. Each value was further normalized to the value for the control (DMSO) sample on the same plate.

Cells from the GFP-GR-expressing cells line, 3617 (Walker et al., 1999 ) were plated in 96 well plates at 15000 cells per well and grown overnight in DMEM medium containing 10% charcoal stripped serum (Hyclone, Logan, UT) without tetracycline to allow the expression of GFP-GR. Cells were then treated with vehicle control, 100nM Dex or 100nM Dex combined with 1nM Nocodazole and 1μM Jasplakinolide for 30 minutes. Upon treatment, cells were fixed with 4% paraformaldehyde in PBS for 10 min, washed 3 times with PBS and counterstained with DAPI. After 3 final washes with PBS cells were stored in PBS at 4°C or imaged immediately. A PerkinElmer (Waltham, MA) Opera QEHS High-Content Screening platform was used for fully automated confocal collection of images. This system employed a 40× water immersion objective lens, laser illuminated Nipkow disk, and cooled CCD cameras to digitally capture high-resolution confocal fluorescence micrographs (300 nm pixel size with 2 × 2 camera pixel binning).

A PerkinElmer (Waltham, MA) Opera QEHS High-Content Screening platform was used for fully automated confocal collection of images. This system employed a 60x water immersion objective lens, laser illuminated Nipkow disk, and cooled CCD cameras to digitally capture high-resolution confocal fluorescence micrographs (300 nm pixel size with 2x2 camera pixel binning). An image analysis pipeline was customized using the Columbus software (PerkinElmer) to automatically segment the nucleus using the DAPI channel and then construct a ring region (cytoplasm) around the nucleus mask for each cell in the digital micrographs. As previously described (Stavreva et al., 2012a (link)) the GR intensity of the 3617 cells (GFP-GR) or the immunoassayed 33Ap1C9 cells was calculated for both nucleus and the cytoplasm and the measurements per well were averaged and normalized to the control treatment.