| |
|
|
|
|
|
|
|
|
Multiparameter Cytotoxicity BioApplication and HCS Reagent Kit |
|
|
DescriptionAn essential part of the drug discovery/approval process is determining the toxic effects of compounds that are potential drugs. Following a toxic insult, cells may respond with changes in size and/or morphology depending on the cell type and the compound. Loss of cell membrane integrity is another common phenotypic feature of cytotoxicity. Some toxins can interfere with the cell’s functionality by affecting the physiology of organelles such as lysosomes and endosomes, or by causing an increase in the number of lysosomes, as in the case of phospholipidosis. The Multiparameter Cytotoxicity BioApplication and HCS Reagent Kit provides users with the ability to rapidly acquire information about changes to the following cellular properties: (1) nuclear morphology/size, (2) cell membrane permeability, (3) lysosomal mass, and (4) cell density (number of cells per field). The Kit contains a cocktail of fluorescent dyes that allows one to measure these cellular properties of interest simultaneously. The BioApplication automatically identifies objects based on a fluorescent nuclear stain (Channel 1). Nuclear size, morphology and cell density measurements are made on identified nuclei in this channel. The BioApplication then measures nuclear intensity in Channel 2 (Green- membrane permeability indicator) and the intensity of lysosomal stain in the cytoplasmic region in Channel 3 (Red). The BioApplication is also capable of automatically setting upper and lower thresholds for nuclear morphology/size, cell membrane permeability and lysosomal mass from user-configurable reference wells (e.g., untreated / negative control wells). These upper and lower thresholds are then used to automatically calculate percentage (indices) of cells in each well that are outside of these thresholds. The cell density per field in the reference wells is also measured and used to calculate a cell density index for experimental wells. The BioApplication reports a Cytotoxicity Index, which is the maximal of nuclear morphology index, membrane permeability index, lysosomal mass index and cell density index, as a convenient single-number indicator of cell health. The use of the Multiparameter Cytotoxicity BioApplication in conjunction with the Multiparameter Cytotoxicity HCS Reagent Kit to determine the cytotoxicity of valinomycin is shown below. HepG2 cells were plated and cultured overnight. The cells were then treated with varying concentrations of valinomycin for 24 hours. Cells were stained, fixed, and scanned in an ArrayScan HCS Reader. Figure 2 shows the effect of exposing HepG2 cells to 100 mM valinomycin for 24 hours. 
Figure 3 shows the dose response to varying concentrations of valinomycin on nuclear morphology/size, cell membrane permeability, and lysosomal mass in HepG2 cells. Drug compounds are primarily targeted to the liver and kidney for metabolism and excretion. Hence, this assay was developed in cell types and/or cell lines that have been derived from these organs. The cells that could be used for this assay include, but are not limited to, rat primary hepatocytes, HepG2, BHK and MDCK cells. Prepared cells can be analyzed using the fully automated Thermo Scientific Cellomics ArrayScan HCS Reader with the Multiparameter Cytotoxicity BioApplication software, affording automated plate handling, focusing, image acquisition, analysis. quantification and data storage.
Features
|
|
|
|
|
|
|
|
|
|
Home 
Products 
top