Fluorescence Cell-Based/Microorganism Screen to Determine Compound Effect on T. cruzi in NIH3T3 Cells
Ana Rodriguez,NYU,Ana.Rodriguez@nyumc.org,212-263-6757(Office),212-263-6589(lab),New York University School of Medicine Department of Medical Parasitology 341 E. 25th St. New York NY 10010 ..more
BioActive Compounds: 27
Primary Collaborators(and laboratory where this assay was performed):
Ana Rodriguez,NYU,Ana.Rodriguez@nyumc.org,212-263-6757(Office),212-263-6589(lab),New York University School of Medicine Department of Medical Parasitology 341 E. 25th St. New York NY 10010
Keywords: Trypanosoma cruzi, Chagas disease
Assay Overview: The aim of the assay is to determine the extent of compound-mediated inhibition of T. cruzi replication and method of action of the compound. Fifty thousand NIH3T3 cells were seeded on sterile glass coverslips in 12-well plates and allowed to adhere overnight. T. cruzi were then harvested and 5,000,000 active trypomastigotes were co-cultured with the NIH3T3 cells for 2 hours. Cells were washed, and those co-cultured cells were incubated with compounds for 4 days. Cells were fixed, stained with DAPI and anti-T.cruzi antibodies, and imaged using a fluorescent microscope.
Expected Outcome: Addition of compounds will suppress T. cruzi replication within co-cultured cells. Static compounds will suppress T. cruzi replication, but amastigote morphology will not be altered. Lytic compounds will produce a diffuse fluorescent staining with anit-T.cruzi antibody in the cytosol.
Immunofluorescence assay to determine lytic or static activity of compounds on Trypanosoma cruzi replication:
Fifty thousand NIH3T3 cells were seeded on sterile glass coverslips in 12-well plates and allowed to adhere overnight. Five million T. cruzi parasites were added (MOI 100:1) and allowed to infect for 2 h in DMEM+2% FBS and Pen-Strep-Glut. Parasites were rinsed out three times with PBS and compounds were added at 10 times their IC50 (as determined in AID 2044 and AID 2294). Infected cells were further incubated for four days and fixed for 15 min with 4% paraformaldehyde.
Fixed cells on coverslips were rinsed with PBS, permeabilized for 15 min in PBS with 0.1% Triton X-100. After blocking for 20 min in PBS with 10% goat serum, 1% bovine serum albumin, 100 mM glycine and 0.05% sodium azide, cells were incubated for 1 h at room temperature with a polyclonal rabbit anti-T. cruzi (gift from Dr B. Burleigh, Harvard School of Public Health, Boston, MA) at 1:2000 dilution. After rinsing, an Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (Molecular Probes, Invitrogen) was added for 1 h at a 1:800 dilution. DNA was stained with DAPI and coverslips were mounted with anti-fade mounting media. Images were taken using an inverted Olympus IX70 microscope with a 60 oil objective.
The aim of the assay is to determine the method of action of the compound and the extent of inhibition of T. cruzi replication. To quantitate relative inhibition of parasite replication, 50 infected cells were counted for each compound. The number of infected cells with more than 4 amastigotes (where the parasite has invaded and is actively replicating) was divided by the total number of infected cells counted. As observed by immunofluorescence, all compounds inhibited T. cruzi replication compared to control, but the parasite maintained defined amastigote bodies. Therefore, the method of action was determined as static. No diffuse staining in the cytosol of host NIH3T3 cells was observed in any case, suggesting no lysis of the amastigotes (as described in PMID 19238193). Therefore, none of the compounds were categorized as lytic.
Images of T.cruzi-infected NIH3T3 cells were examined manually and scored for appearance, noting the presence of defined amastigote bodies (indicates static effect), diffuse staining in the cytosol of host cells (indicates lytic effect) and the percent of infected cells with more than 4 amastigotes (indicates the parasite is actively replicating).
The neutral control was DMSO only, with no compound.
DMSO control yielded 42% of T. cruzi infected cells with replication (> 4 amastigotes)
Compounds were tested at the concentrations indicated.
Percent inhibition of replication of the intracellular parasite was calculated as:
100 - (100 * (% infected cells with replication at dose/ % infected cells with replication at DMSO))
The Percent Inhibition value.
Activity_Outcome = 1 (inactive)
PubChem_Activity_Score < 50
Activity_Outcome = 2 (active)
PubChem_Activity_Score >= 50
Categorized Comment - additional comments and annotations
Data Table (Concise)