To optimize image acquisition during assay development and HTS, a "z-stack" of images was taken using the Opera software to determine the optimum focal plane for imaging, but more specifically for assay reproducibility.
The instrument settings used for image acquisition also affected assay quality, and were improved considerably by optimizing the microscope's autofocus and scanning times, and the degree of magnification used to scan the wells of 96- versus 384-well plates.
Moreover, the computational analysis that follows image acquisition required by these assays can be complicated and often requires an intermediate step of cell tracking (as for assays shown in Fig. 1c f), and the extraction of indirect parameters (acceleration, Cx acceleration index) to ascertain CIL.
Nonetheless, experimental variables that affect high-quality HCS protocols, such as sample preparation, assay strategy, image acquisition and image analysis, have yet to be optimized for any organism [41].
Genotypes were determined by PCR assay after image acquisition (immunohistochemistry) or from larval tail clips after in situ hybridization.
The image acquisition and analysis in our assay is simple and straightforward as only a standard fluorescence microscope system and image analysis software are necessary.
The simplification of the assay protocol, image acquisition, and data analysis allows for large numbers of compounds to be screened in a relatively short period of time.
RP carried out the amorphous cellulose, CBD and CBD-FITC production, made the adsorption assays and the image acquisition, participated in the conception of this work and manuscript writing.
Here, we describe an automated PR assay, including sample preparation, automated image acquisition, and a computational image analysis pipeline using open-source software in order to convert the PR assay for HIV neutralization into a high-throughput, high-content assay.
