This protocol describes a high throughput colorimetric assay in the three lifecycle stages of Trypanosoma cruzi to identify new drug candidates for Chagas disease. This can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner. Demonstrating the procedure will be Romina Manarin, a lab cell culture technician from the laboratory of Pamela crib.
Begin by culturing beta galactosidase expressing Trypanosoma cruzi epimastigotes in a T-25 tissue culture flask and incubate at 28 degrees Celsius for growing them axenically. Using a Neubauer chamber, quantify parasite growth before subculturing. For maintaining the cultures in a log phase, subculture every 48 to 72 hours in five milliliters of LIT medium supplemented with 10%FCS and geneticin in sulfate at a final concentration of 200 micrograms per milliliters.
Then securely close the cap before incubating at 28 degrees Celsius in a vertical position. From the log phase culture, make a suspension of epimastigote in LIT medium supplemented with geneticin and sulfate at the final concentration of 200, 000 cells per milliliter epimastigote per milliliter. After dispensing 100 microliters of suspension per well of a 96 well plate, makeup final volume of well 200 microliters with medium.
Make the Vero cell suspension in DMEM supplemented with 2%FCS at the final concentration of 100, 000 cells per milliliter. Seed 100 microliters of the suspension per well in a 96 well tissue culture plate. Incubate the cells overnight for adherence and the next day, observe the cells under the microscope.
The next day, rinse the Vero cell monolayer three times with 100 microliters of sterile PBS. Add previously obtained metacyclic trypomastigotes. Add a multiplicity of infection or MOI of 10-1 in 100 microliters of DMEM supplemented with 2%FCS per well and incubate for six hours as demonstrated previously.
At the end of the incubation, wash the plate twice with PBS and add 100 microliters of DMEM without phenol red supplemented with 2%FCS. After making the Vero cells suspension in DMEM add the final concentration of 1 million cells per milliliter. Seed 800, 000 cells in five milliliters of the medium in T-25 flasks and incubate overnight for cell adherence.
Once the flask is rinsed with PBS, add trypomastigotes, add an MOI of 10-1 in five milliliters of DMEM supplemented with 2%FCS and incubate overnight as demonstrated previously. At the end of incubation, wash the flask twice with PBS. Add five milliliters of fresh DMEM supplemented with 2%FCS and incubate for four days.
At the end of the incubation, after observing the supernatant for trypomastigotes presence under an optic microscope. Quantify the trypomastigotes using a Neubauer chamber. Collect the supernatant in a 15 milliliter tube and centrifuge at 7, 000 times G for 10 minutes at room temperature.
Then discard the supernatant and resuspend the pellet in DMEM without phenol red supplemented with 2%FCS to obtain a concentration of 1 million trypomastigotes per milliliter. Seed 100 microliters of the trypomastigote suspension per well in a 96 well plate. Add benznidazole solution to epimastigotes, Vero cells with amastigotes, or trypomastigotes as described in the text manuscript.
Then incubate the epimastigotes, set 28 degrees Celsius for 72 hours and the trypomastigotes or infected Vero cells with amastigotes for 24 hours at 37 degrees Celsius and 5%carbon dioxide. For the colormetric assay, set up the blank wells by adding 100 microliters of PBS or corresponding medium. Next, add 40 microliters of CPRG substrate solution and 10 microliters of the detergent solution to each well for obtaining a final concentration of 200 micromolar CPRG and 0.1%detergent in a final volume of 250 microliters in each well.
After incubation, at 37 degrees Celsius for two hours, measure absorbance at 595 nanometers using a microplate spectrophotometer. In the software, create a new protocol by selecting absorbance as the detection method and end point as the read type, then click okay. Next, add a read step.
Type the selected wavelength and click okay. In the plate layout section mark the wells to be read, click okay, and click on read plate, wait for the values to appear on the screen and export them to a spreadsheet to analyze the results and calculate the absorbance values using subtraction as described in the manuscript. In statistical analysis software, plot the concentration of BZN versus the absorbance at 595 nanometers in the XY table.
Transform the BZN concentrations to logarithmic values by clicking analyze, selecting the transform option, and clicking okay. For obtaining IC50 values, click analyze, and from the XY analysis list select nonlinear regression curve fit, then click okay. Next, go to the parameters window, in the model tab, go to the dose response inhibition group of built-in equations, select log inhibitor versus response variable slope for parameters as the dose response method leave all other tabs at default values and then click, okay.
Click the results section to find the IC50 value, the SD, and the goodness of the fit. Click the graph section to find the XY graph of the logarithmic concentration of the drug versus the absorbance values and ensure that the curve fit is also graphed in a different color. In the present study, the IC50 value obtained for epimastigotes was approximately 20 micromolar, similar to previous studies.
In addition, the results indicated liner beta galactosidase activity of the epimastigotes over time. It is very important to perform at least replicates of each condition tested and minimized errors in volume management.
