Approximately 500 cells from each biopsy were seeded in each well of 1,536-well microtiter plates (PerkinElmer) and incubated overnight. Combinatorial drug library was pin-transferred to the seeded plates as previously described (8 (link)). Plates were incubated for 96 hours, fixed in 4% formaldehyde, washed in PBS containing 0.1% Triton (PBST), and incubated overnight with antibodies (1:200) to S100 (Dako). The plates were then washed twice, incubated with Alexa 488 secondary antibodies (1:1000, Life Technologies) and DAPI 2–16 hours, and washed with PBST. Plates were then imaged using the CellWorX high-throughput microscope (Applied Precision Inc.) and nuclei and cells with S100 staining were counted with the Multi-Wavelength Cell Scoring module of the MetaExpress image analysis software (Molecular Devices). Representative control well images from each sample were manually reviewed to confirm S100 staining corresponded to cells with melanoma morphology. Technical replicates were averaged. Where replicates were available, Z’ scores were calculated (19 (link)), varying from 0.15 for CBRC029 to 0.58 for CBRC056.
Metaexpress image analysis software
Manufactured by Molecular Devices
MetaExpress is an image analysis software designed for analyzing and processing microscopic images. It provides tools for tasks such as cell segmentation, object counting, and fluorescence intensity measurements. MetaExpress is compatible with a variety of microscopy techniques and file formats, allowing users to analyze diverse types of image data.
Automatically generated - may contain errors
2 protocols using metaexpress image analysis software
1
High-Throughput Melanoma Phenotypic Screening
2
High-throughput Apoptosis Screening Assay
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Approximately 250 cells per well for each cell line were seeded in each well of 1,536-well microtiter plates (PerkinElmer) and incubated overnight. Combinatorial drug library was prepared as 300X stocks in 384 well plate and 20nL pin-transferred to 1,536-well plates. Plates were incubated for 72 hours and fixed in 4% formaldehyde, washed in PBS containing 0.1% Triton, and incubated overnight with antibodies (1:500) to cleaved PARP (above). The plates were then washed twice, incubated with Alexa 647 secondary antibodies (1:1000, Life Technologies) and DAPI 2–16 hours, and washed with PBST. Plates were then imaged using the Cellworx high-throughput microscope (Applied Precision Inc.) and counting nuclei and cells with cPARP staining with the Multi-Wavelength Cell Scoring module of the MetaExpress image analysis software (Molecular Devices). Z’ values were between 0.8–0.9 for most cell lines.
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