Inhibition of intracellular CAI/72 strain of Trypanosoma cruzi in bovine embryo skeletal muscle cells: high content imaging Measured in Cell-Based System Using Imaging - 2138-07_Inhibitor_SinglePoint_DryPowder_Activity
Assay Overview: The assay detects the effects of compounds on intracellular T. cruzi amastigotes. Bovine Embryo Skeletal Muscle (BESM) cells are infected with CA-I/72 clinical strain of T.cruzi in a clear bottom 96-well plate. Extracellular T. cruzi is washed away and varying concentrations of compound are added to the well and incubated for 72 hrs. Cells are fixed and stained with a nuclear dye Hoechst and the number of T. cruzi vs. mammalian host cell nuclei are imaged, categorized by size, and counted using the IN Cell Analyzer 2000. ..more
BioActive Compounds: 28
Keywords: Trypanosoma cruzi, amastigotes, high content screening, Chagas Disease
Assay Overview: The assay detects the effects of compounds on intracellular T. cruzi amastigotes. Bovine Embryo Skeletal Muscle (BESM) cells are infected with CA-I/72 clinical strain of T.cruzi in a clear bottom 96-well plate. Extracellular T. cruzi is washed away and varying concentrations of compound are added to the well and incubated for 72 hrs. Cells are fixed and stained with a nuclear dye Hoechst and the number of T. cruzi vs. mammalian host cell nuclei are imaged, categorized by size, and counted using the IN Cell Analyzer 2000.
Expected Outcome: Compounds significantly suppressing luminescence, and therefore b-galactosidases expression will be identified as hits in the screen. Compounds that inhibit luminescence activity may kill T. cruzi, inhibit T. cruzi invasion or inhibit development of the parasite within the host cell. Compounds that are toxic to the host cell will be excluded in secondary assays.
Microtiter-based 72 hour screen against T. cruzi amastigotes in host BESM cells
The screen employs the IN Cell Analyzer 2000 (GE Healthcare Life Sciences), a flexible, modular, cellular and subcellular imaging system for fast, automated imaging of fixed and live cells built around an automated epifluorescence-based microscope and image acquisition software. The screening is performed in sterile and fluorescence transmissible 96 wells-cell culture plates. Each well is seeded with 1000 BESM cells (Bovine Embryo Skeletal Muscle Cells) allowed to attach for several hours. The culture medium (RPMI 1640 with 5 % FBS) is then replaced with fresh medium containing 1000 T. cruzi trypomastigotes (Wild type CA-I/72 T. cruzi was isolated from an Argentinean chronic chagasic patient ) per well. Sixteen hours post-infection, cells are washed once to remove extracellular parasites, and maintained in medium containing serial dilutions of compounds for 72h at 370C. Cells are then washed once with PBS, fixed for 2 hours with 4% paraformaldehyde, and washed 1 time to remove the fixative. Then 50 microl of mounting medium containing a DNA fluorescent dye (i.e. DAPI or Hoechst) are added per well. The plates are kept in the dark at 4oC until scanned in the IN Cell Analyzer with a 10X objective, and the images are collected with appropriate fluorescent excitation/emission filters
At least 100 infected cells are recorded per well. The ratio of T. cruzi amastigotes per infected cell is calculated in untreated controls and compared to those obtained for the different compounds and conditions tested. A significant reduction in the E/C ratio indicates inhibition of parasite growth rate. This information provides a quantitative measure of growth inhibition during the first 72h post-infection.
In addition to IC50 values, IC90 and IC99 are provided for some active compounds. Also included but not used for calculation are Host cell toxicity measurements.
Compounds that did not achieve sufficient activity were listed with IC50 equal to the greatest tested concentration where applicable and should be interpreted as having an IC50 > 20000nM.
Compounds that had IC50 less than 3 times the control compound, Benznidazole, were labeled active. Those samples that had one test active and an other inactive were labeled inconclusive.
Pubchem Activity Score was calculated by dividing the IC50 of the control compound, Benznidazole, by the compound's average IC50 and multiplied by 100. Larger numbers are more active and lower numbers are less active.
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* Activity Concentration.
Data Table (Concise)