4 6 diamidino 2 phenylindole (dapi)

Microscopic imaging-based drug screening with breast cancer cell lines was performed using an Olympus scan^R integrated high content imager and image analysis suite (Olympus-SIS). AZD1775 and AZD6738 was tested with six 3-fold dilutions starting from 10 µM and 20 µM as the highest concentration, respectively. Each 384-well sample well was imaged with a 10 × objective using specific filter sets for DAPI (Semrock, Inc). The effect of the drugs on cell numbers as indicator of cell viability was assessed by comparing cell counts measured in DMSO (negative controls). The DNA counterstaining of the cells was performed according to the following protocol. First the culture medium was aspirated carefully from each well and the cells were fixed with 2% paraformaldehyde (Sigma-Aldrich) in PBS for 15 min at room temperature. Cells were then washed once for 5 min with PBS. Cell permeabilized was done with 0.3% Triton-X100 in 20 µL of PBS for 15 min at room temperature, followed by PBS wash. DAPI (4′,6-diamidino-2-phenylindole) DNA counterstaining was performed for 1 hr at room temperature, followed by washing with PBS.

Images of internalized and extracellular bacteria were acquired using a widefield epifluorescent Nikon Ti2-E inverted microscope with Nikon SR Plan Apo IR 60xAC WI, NA = 1.27. Fluorescence was excited with the SPECTRA X light engine^® (Lumencore Inc, Beaverton, OR, USA) and collected with DAPI (Exc. 379-405 nm, Em. 414-480 nm), FITC (Exc. 457-487 nm, Em. 503-538 nm), TRITC (Exc. 543-566 nm, Em. 582-636 nm) and Cy5 (Exc. 590-645 nm, Em. 659-73 6nm) filter cubes, all from Semrock. Images were acquired using Nikon DS-Qi2 CMOS controlled with NIS-Elements (v. 5.21.02). Multiple stage positions were collected using a Nikon motorized stage. For each data set, 3x3 20X magnification images were first collected, including z-stacks, allowing them to cover a wide area of the sample. From these images, multiple areas of interest were imaged at 60X and deconvolved using NIS-Elements (v 5.21.02) to remove out-of-focus light. After analysis of the deconvolved images, representative images were selected. Image gamma, brightness, and contrast were adjusted using NIS-Elements Viewer.