Imagexpress micro 4 high content screening system

The proliferation of HCC cells was determined by EdU assay. After HCC cells (4,000 cells/well) were seeded in 96-well plates and incubated for 24 h, cells were exposed to concentrations of ESO (0, 62.5, 125, and 250 μg/ml) for 24 h and incubated with 10 μM EdU (APExBIO, Houston, United States) for another 2 h. Then the cells were fixed with 3.7% formaldehyde for 15 min and cell nuclei were stained with Hoechst 33,342. Eventually, EdU-positive cells were observed and photographed under a photographed at a magnification of ×100 with an ImageXpress - Micro 4 High-Content Screening system (Molecular Devices, LLC, CA, United States). The EdU-positive cells (%) = The count of red EdU/The count of blue Hoechst 33,342 × 100.

EdU is a thymidine analog that can be incorporated into replicating DNA for the detection of cell proliferation (Salic and Mitchison, 2008 (link)). In this study, NSCLC cells were seeded into 96-well plates (4 × 10³ cells/well) and incubated for 24 h before treatment with different concentrations of SOL (0, 50, 100, or 200 μg/ml) for 24 h. Cells were then incubated for 2 h with 50 μM EdU (#K1075, APE×BIO, TX, United States) before fixation with 4% formaldehyde (#1810473, Biosharp, Hefei, China) for 30 min. EdU Click buffer was then added for 30 min and cells were stained with Hoechst 33342 for 30 min. EdU-positive cells were imaged with an ImageXpress Micro 4 High-Content Screening System (Molecular Devices, CA, United States) at ×100 magnification. Cell proliferation was analyzed as the percentage of viable cells using the following formula: $Cellproliferation\left(\%\right)=\frac{NumberofEdUstainedcells\left(red\right)}{NumberofHoechst33342stainedcells\left(blue\right)}\times 100$

Either an SP8 confocal microscope (Leica), ImageXpress Micro 4 High Content Screening System (Molecular Devices) with an Andor SDK3 camera, CKX53 inverted microscope with an SC100 digital camera (Olympus), or a BX51 upright microscope (Olympus) with a PCO.Panda 4.2 digital camera were used for image acquisition. All images were taken using 16-bit cameras. Varying lens parameters and acquisition settings were used, as follows: