The myImageAnalysis Project: A Web-Based Application for High-Content Screening

Abstract

A major challenge faced by screening centers developing image-based assays is the wide range of assays needed compared to the limited resources that are available to effectively analyze and manage them. To overcome this limitation, we have developed the web-based myImageAnalysis (mIA) application, integrated with an open database connectivity compliant database and powered by Pipeline Pilot (PLP) that incorporates dataset tracking, scheduling and archiving, image analysis, and data reporting. For system administrators, mIA provides automated methods for managing and archiving data. For the biologist, this application allows those without any programming or image analysis experience to quickly develop, validate, and share results of complex image-based assays. Further, the structure of the application within PLP allows those with experience in PLP programming to easily add additional analysis tools as required. The tools within mIA allow users to assess basic (cell count, protein per cell, protein subcellular localization) and more advanced (engineered cell lines analysis, cell toxicity) biological image-based assays that employ advanced statistics and provides key assay performance metrics.

Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells

“…To visualize directly the population of fluorescing cells, assess the effect of vector length on transfection by a separate method, and test multiple concentrations of vector doses, we used fluorescence microscopy and an automated method [21] to quantify GFP knockdown. We transfected each of the unlabeled vectors at 4, 40, or 400 fmol into HeLa-GFP cells.…”

“…For each experiment, “no DNA added” and pT7 (a plasmid that encodes the same shRNA as the other vectors, but under a T7 promoter that does not express in mammalian cells), served as negative controls. From high resolution fluorescent images, cell area was calculated and GFP fluorescence per cell was quantified (S1 Fig) [21]. This process was done for the entirety of one visual field per well of a 96-well plate, with the total cells read ranging from 680 to 6,407 (depending on the number of cells visible in each field) for each transfection condition (Fig 5).…”

“…Photographs of fields of cells from several wells from each transfection were taken. Images were analyzed using myImageAnalysis [21] to quantify GFP fluorescence and size for each cell, encompassing several thousand cells in each transfection. Data were plotted using a Tukey box plot where each outlier cell, defined by having a GFP per cell area more than 1.5 x interquartile range above the 75 th percentile, is denoted by a circle.…”

CUDC-101, a Novel Inhibitor of Full-Length Androgen Receptor (flAR) and Androgen Receptor Variant 7 (AR-V7) Activity: Mechanism of Action and In Vivo Efficacy

“…In a parallel project, we embarked on a small library screen of FDA approved drugs to establish classes of bioactive molecules inhibiting AR signaling, which may be useful to treat CRPC [18]. Enrichment analysis of active compounds showed that histone deacetylase inhibitors (HDACi) were most active (manuscript in preparation).…”

“…According to a number of recent reports [10, 11], the accumulation in the recurring tumor of the carboxyl terminal truncated and constitutively active AR variant (AR-V), AR-V7 [12-17] is an important contributor to resistance to second generation ADT. In a parallel project, we embarked on a small library screen of FDA approved drugs to establish classes of bioactive molecules that inhibit AR and AR-V7 signaling, and thus may be useful to treat CRPC [18]. One class of compounds identified with this approach (manuscript in preparation) consisted of histone deacetylase inhibitors (HDACi), which lead to testing commercially available agents with the same mechanism of action.…”