Scanr high content screening station

The surface marker expression of dendritic cells was analyzed by microscopy after staining with anti-human HLA-DR Alexa 488 and anti-human CD86 Alexa 647 (both from Biolegend, London, UK) and putting the cells in a 96-well black clear bottom plate (BD Bioscience, Heidelberg, Germany). The images were acquired using an Olympus Scan^R High Content Screening Station based on a IX81 stage and using either the 20x 0.45 LUCPLFLN (quantitation) or 40x 0.9 UPLSAPO (close up images) objectives, and Scan^R Acquisition software v. 2.6.2. Analysis of the images was done using Scan^R Analysis software v. 2.6.2. Acquisition and analysis settings were identical for each well. For the analysis, after background subtraction, the segmentation mask used to identify the cells had an intensity threshold for either the Alexa 488 (HLA-DR) and Alexa 647 (CD86) signals, respectively, and used a watershed transform to limit the number of doublets. The Alexa 488 and Alexa 647 intensities, respectively, for all pixels of each cell were summed and defined as the total intensities for each channel, respectively. The means of these total intensity values were then calculated for each well and fluorochrome.

Brightfield and fluorescent images were obtained using an automated fluorescence microscope (Olympus ScanR High Content Screening Station) using the 20x UPLSAPO NA 0.75 objective. Cell images were classified according to cell viability (CV), programmed apoptosis (AP), autophagy (AG), or cell death (CD). In total, 7500 images were analyzed using the Fiji program (v.2.0.0) and the Trainable Weka Segmentation plugin (v.3.2.33): The Weka plugin within ImageJ is an open-source platform for biological-image analyses that combines a collection of machine learning algorithms with a set of selected image features to produce pixel-based segmentations [24 (link),25 (link)]. All of the experiments were performed in triplicate and the data are presented as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) and Student’s t-test were used for comparisons of each group. P-values less than 0.05 were considered statistically significant and are indicated with asterisks.