We thank Dr. Buckner for kindly providing the pLacZ plasmid. This work was supported by Agencia Nacional de Promoci&#243;n Cient&#237;fica y Tecnol&#243;gica, Ministerio de Ciencia e Innovaci&#243;n Productiva from Argentina (PICT2016-0439, PICT2019-0526, PICT2019-4212), and Research Council United Kingdom [MR/P027989/1]. Servier Medical Art was used to produce Figure 1 (https://smart.servier.com).

NOTE: An overview of the entire experimental design is depicted in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63210/63210fig01.jpg" /> 
Figure 1: Overview of the in vitro screening assay of Trypanosoma cruzi Dm28c/pLacZ line using CPRG as a substrate for the colorimetric reaction. The assay consists of seeding the parasites (1), incubating them with BZN (2 and 3), and then adding the colorimetric substrate (4). When parasites are lysed, &#946;-galactosidase is released and cleaves CPRG to chlorophenol red; this change in color can be measured spectrophotometrically (5). Data can be analyzed in statistical analysis software to obtain the half inhibitory concentration (IC50) of BZN. Abbreviations: CPRG = chlorophenol red &#946;-D-galactopyranoside; BZN = benznidazole. <a href="https://www.jove.com/files/ftp_upload/63210/63210fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Preparation of stock solutions
<ol>
	<li>Preparation of media and solutions
	<ol>
		<li>Hemin solution (Supplemental Table S1)
		<ol>
			<li>Add all the components to a 50 mL centrifuge tube in the order given in the recipe and homogenize by inversion several times.</li>
			<li>Sterilize by filtration through a 0.22 &#181;m filter.</li>
			<li>Prepare 1 mL aliquots in 1.5 mL microcentrifuge tubes and keep them at -80 &#176;C until use.</li>
		</ol>
		</li>
		<li>Liver Infusion Tryptose (LIT) Medium (Supplemental Table S1)
		<ol>
			<li>Weigh all the components and stir to homogenize at room temperature in a 1 L beaker containing at least 700 mL of distilled water.</li>
			<li>Adjust the pH to 7.2 and top up the volume to 900 mL in a 1 L graduated cylinder with distilled water; sterilize by filtration or autoclaving (121 &#176;C for 20 min).</li>
			<li>Supplement the medium by adding 100 mL of fetal calf serum (FCS), (10% FCS, sterile and heat-inactivated at 56 &#176;C for 45 min), 20 mL of 40% sterile glucose solution (sterilized by autoclaving, 121 &#176;C for 20 min), and 5 mL of hemin solution (final concentration 5 &#181;M) to 900 mL of LIT medium.</li>
		</ol>
		</li>
		<li>Prepare Dulbecco&#39;s Modified Eagle Medium (DMEM) from the powder following the manufacturer&#39;s instructions.</li>
		<li>Phosphate-buffered saline (PBS) (Supplemental Table S1)
		<ol>
			<li>Dissolve all solid components by stirring the solution at room temperature in a 1 L beaker.</li>
			<li>Adjust the pH to 7.2, level up to 1 L in a 1 L graduated cylinder with distilled water, and sterilize by filtration or autoclaving (121 &#176;C, 20 min).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Benznidazole (BZN) stock solutions and dilutions 
	NOTE: The range of BZN concentration used in this work was 2.5 to 80 &#181;M.
	<ol>
		<li>Prepare a stock solution of 1 M BZN by dissolving 13 mg of the drug in 50 &#181;L of dimethylsulfoxide (DMSO). Under aseptic conditions, prepare serial dilutions from this 1 M BZN stock solution at twice the final desired concentration (2x solutions) in a final volume that is adequate for the number of wells to be assayed. 
		NOTE: Calculate for 100 &#181;L per well with an excess of 10-20%. The BZN stock solution and all BZN dilutions must be prepared immediately before use in the assay due to the low solubility of the drug in the medium.</li>
		<li>Prepare 2x BZN dilutions of 160, 80, 40, 20, 10, and 5 &#181;M.
		<ol>
			<li>Dilute 1 M BZN stock solution at a 100-fold dilution (10 &#181;L of 1 M BZN + 990 &#181;L of medium) to obtain a 10 mM solution in the appropriate medium used for each life cycle stage of T. cruzi. Mix continuously to homogenize the suspension.</li>
			<li>Dilute 10 mM BZN solution to prepare 320 &#181;M BZN in the appropriate medium: 32 &#181;L of 10 mM BZN + 968 &#181;L of medium. Mix continuously to homogenize the suspension.</li>
			<li>Dilute 320 &#181;M BZN 2-fold to obtain a concentration of 160 &#181;M (500 &#181;L of 320 &#181;M BZN + 500 &#181;L of medium). Mix continuously to homogenize the suspension. Repeat this 2-fold dilution with each resulting solution to obtain 80, 40, 20, 10, and 5 &#181;M solutions.</li>
			<li>Dilute DMSO 1,000-fold in the appropriate medium for use as untreated control (100% survival control). 
			NOTE: Epimastigotes tolerate up to a 100-fold dilution of DMSO, whereas Vero cells tolerate only up to a 1,000-fold dilution of DMSO. If necessary, a death control with 50% DMSO can be included as the 0% survival condition.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Substrate solution
	<ol>
		<li>Dissolve CPRG at 1 mM concentration in distilled water. For a 96-well plate, add 2.4 mg of CPRG to 4 mL of water. 
		NOTE: CPRG solution must be prepared immediately before the assay.</li>
	</ol>
	</li>
	<li>Lysis solution
	<ol>
		<li>Prepare a 2.5% v/v solution of nonionic, non-denaturing detergent 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (see the Table of Materials) in 1x PBS. Prepare 1 mL of the solution per 96-well plate immediately before the assay.</li>
	</ol>
	</li>
</ol>
2. Parasite culture preparation
<ol>
	<li>Epimastigote preparation 
	NOTE: T. cruzi Dm28c/pLacZ line13 is used throughout this report.
	<ol>
		<li>Grow the &#946;-galactosidase-expressing T. cruzi epimastigotes axenically at 28 &#176;C in cell culture flasks with a growth area of 25 cm2 (T-25 flasks). Maintain the cultures in log phase by subculturing every 48-72 h (in 5 mL) in LIT medium supplemented with 10% FCS (Supplemental Table S1) and geneticin sulfate (G418) at a final concentration of 200 &#181;g/mL. Quantify parasite growth by cell counting in a Neubauer chamber before subculturing. Securely close the cap and keep the culture flask (not vented) at 28 &#176;C in a vertical position. 
		NOTE: G418 ensures pLacZ plasmid selection and maintenance. Log phase cultures have an epimastigote concentration of 1-5 &#215; 107 parasites/mL for the Dm28c/pLacZ line.</li>
		<li>Prepare a suspension of 2 &#215; 105 epimastigotes/mL from a log phase culture in LIT supplemented with G418 antibiotic. Dispense 100 &#181;L of the epimastigote suspension per well (20,000 epimastigotes in 100 &#181;L of LIT) of a 96-well microplate and make up the final volume to 200 &#181;L per well with the medium.</li>
	</ol>
	</li>
	<li>Amastigote preparation
	<ol>
		<li>Use spontaneous metacyclic trypomastigotes obtained from an aged epimastigote culture (for 7 days in this protocol) to perform an initial infection in a T-25 flask with 2 &#215; 105 Vero cells seeded previously in DMEM supplemented with 2% FCS.
		<ol>
			<li>Count the number of metacyclic trypomastigotes in a Neubauer chamber, infect the Vero cell monolayer with a multiplicity of infection (MOI) of 10 in 5 mL of DMEM with 2% FCS, and incubate at 37 &#176;C and 5% CO2 for 16 h. Wash the remaining trypomastigotes by removing the medium from the flask with a 5 mL sterile pipette, then add 5 mL of 1x PBS and aspirate. Finally, add 5 mL of DMEM with 2% FCS and incubate under the same conditions.</li>
			<li>Use the trypomastigotes emerging from the infected Vero cell monolayer to maintain the infection in T-25 flasks&#160;with 2 &#215; 105 Vero cells in DMEM with 2% FCS, generating a new infected bottle every week. 
			NOTE: After 5-7 days, trypomastigotes start to emerge and are visible in the supernatant. Do not add G418 to the trypomastigotes used to infect the cells or the infected cells, as the Vero cell line is not resistant to G418.</li>
		</ol>
		</li>
		<li>Prepare a suspension of 1 &#215; 105 Vero cells/mL in DMEM supplemented with 2% FCS and seed 100 &#181;L of the suspension per well in 96-well tissue culture plates (10,000 cells per well). Incubate overnight (12-16 h) at 37 &#176;C and 5% CO2 to ensure cell adherence to the bottom of the wells.</li>
		<li>After the overnight incubation, rinse the Vero cell monolayer three times with 100 &#181;L of sterile 1x PBS. Add T. cruzi Dm28c/pLacZ trypomastigotes (obtained from a previous infection in a T-25 flask, step 2.2.1) at an MOI of 10 in 100 &#181;L of DMEM supplemented with 2% FCS per well (100,000 trypomastigotes per well).</li>
		<li>Incubate the plates for 6 h at 37 &#176;C and 5% CO2. After this incubation period, wash the plates twice with 1x PBS, and add 100 &#181;L of DMEM without phenol red supplemented with 2% FCS. 
		NOTE: After 48 h (2 days post infection), intracytoplasmic amastigotes are visible with an optic microscope. Phenol red, a pH indicator in DMEM and other cell culture media, could interfere with the absorbance measurement of CPRG. If DMEM without phenol red is not available, see alternatives mentioned below in section 3.2.1.</li>
	</ol>
	</li>
	<li>Trypomastigote preparation
	<ol>
		<li>Prepare a suspension of 1 &#215; 106 Vero cells/mL in DMEM supplemented with 2% FCS and seed 800,000 cells in 5 mL of the medium in T-25 flasks. Incubate overnight (12-16 h) at 37 &#176;C and 5% CO2 to ensure cell adherence. 
		NOTE: For a T-75 flask, seed 2 &#215; 106 cells in a final volume of 15 mL.</li>
		<li>After incubation, rinse two times with 3 mL of sterile 1x PBS. Add T. cruzi Dm28c/pLacZ trypomastigotes at an MOI of 10 in 5 mL of DMEM with 2% FCS (8 &#215; 106 trypomastigotes for a T-25 flask). 
		NOTE: For a T-75 flask, add 20 &#215; 106 trypomastigotes in a final volume of 15 mL of DMEM with 2% FCS.</li>
		<li>Incubate overnight (12-16 h) at 37 &#176;C and 5% CO2. Wash the flask twice with 3 mL of 1x PBS, and add 5 mL of fresh DMEM supplemented with 2% FCS. Incubate at 37 &#176;C and 5% CO2 for four days.</li>
		<li>Check the supernatant for trypomastigotes under an optic microscope. Quantify the trypomastigotes by counting them in a Neubauer chamber. Collect the supernatant in a 15 mL tube and centrifuge at 7,000 &#215; g for 10 min at room temperature.</li>
		<li>Discard the supernatant and resuspend the pellet to obtain a concentration of 1 &#215; 106 trypomastigotes/mL in DMEM without phenol red supplemented with 2% FCS. Seed 100 &#181;L of the trypomastigote suspension (100,000 trypomastigotes per well) in a 96-well plate. 
		&#8203;NOTE: If DMEM without phenol red is not available, see alternatives below in section 3.2.1.</li>
	</ol>
	</li>
</ol>
3. &#946;-galactosidase assay
NOTE: Quantitation of &#946;-galactosidase activity is used as an indirect way of determining the number of parasites. It is expected that growth will be inhibited in the presence of a trypanocidal compound, leading to a lower number of parasites compared to the untreated control, which will be reflected in a lower &#946;-galactosidase activity and therefore lower absorbance.
<ol>
	<li>Incubate the parasites with BZN.
	<ol>
		<li>Add 100 &#181;L of corresponding 2x BZN solution per well to reach a final concentration of BZN of 80, 40, 20, 10, 5, and 2.5 &#181;M to 100 &#181;L of epimastigote suspension (from step 2.1), Vero cells with amastigotes (2 days post infection) (step 2.2), or trypomastigotes (step 2.3) in a 96-well plate.</li>
		<li>Incubate the epimastigotes at 28 &#176;C for 72 h, and the trypomastigotes or infected Vero cells with amastigotes for 24 h at 37 &#176;C and 5% CO2. 
		NOTE: Each drug concentration should be evaluated at least in triplicate and include control cultures of epimastigotes, trypomastigotes, and infected Vero cells with DMSO (see step 1.2.2.4).</li>
	</ol>
	</li>
	<li>Colorimetric reaction
	<ol>
		<li>After the treatment incubation period, if infected Vero cells or trypomastigotes are in DMEM with phenol red, replace the medium with 100 &#181;L of 1x PBS to avoid interference. Perform triplicate blank wells containing only 100 &#181;L of corresponding medium (or 1x PBS as appropriate). 
		NOTE: It is not necessary to remove the culture medium for epimastigotes in case of LIT medium or DMEM without phenol red. DMEM with phenol red can still be used; prepare a blank well with DMEM alone to measure the base absorbance and then subtract this value during data analysis (step 3.3.). Schneider&#39;s insect medium, which is colorless, is an alternative for epimastigotes.</li>
		<li>Add 40 &#181;L of CPRG substrate solution and 10 &#181;L of the detergent solution to each well, obtaining a final concentration of 200 &#181;M CPRG and 0.1% detergent in a final volume of 250 &#181;L in each well. 
		NOTE: The CPRG solution and detergent can be added together in a final volume of 50 &#181;L per well.</li>
		<li>Incubate at 37 &#176;C for 2 h and measure the absorbance at 595 nm in a microplate spectrophotometer. 
		NOTE: The expected color change is yellow to reddish-brown upon &#946;-galactosidase enzymatic cleavage (Figure 2A). Incubation time can be extended up to 4 h, and the absorbance spectra of chlorophenol red can be read between 570 and 595 nm with similar curve fittings (Supplemental Figure S1A,B). Incubation for up to 24 h in the presence of CPRG substrate has shown similar curve fittings (Supplemental Figure S1C).
		<ol>
			<li>In a microplate spectrophotometer with a monochromator selector, create a new protocol in the equipment software (Supplemental Figure S2).</li>
			<li>Click Absorbance as detection method | Endpoint as read type | Ok&#160;(Supplemental Figure S2A). Add a Read Step, type the selected wavelength, and click Ok&#160;(Supplemental Figure S2B).</li>
			<li>In the Plate Layout section, mark the wells to be read and click Ok (Supplemental Figure S2C). To read the plate, insert it in the tray and click on Read Plate. Wait for the values to appear on the screen (Supplemental Figure S2D) and export them to a spreadsheet to analyze the results.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Data analysis and media inhibitor concentration (IC50) calculation
	<ol>
		<li>Subtract the blank measured value, corresponding to only LIT medium, 1x PBS, or DMEM with or without phenol red plus the CPRG-detergent solution. When testing the trypanocidal activity of colored compounds, measure the absorbance of additional blank controls with LIT or DMEM with each concentration of drug used and then subtract those values from the absorbance values obtained with the parasites at each concentration. 
		NOTE: Supplemental Table S2 shows typical values obtained in this assay with these media without parasites plus CPRG-detergent solution. The differences before and after adding CPRG are significant but do not interfere with the assay with parasites (Supplemental Table S2).</li>
		<li>In statistical analysis software, plot the concentration of BZN (in &#181;M) versus the absorbance at 595 nm in an xy table. Transform the BZN concentrations to logarithmic values by clicking on the Analyze button, selecting the Transform option | transform the x-values using x=log(x) option, and clicking the Ok button.</li>
		<li>Obtain the IC50 values from the statistical analysis software. 
		NOTE: The IC50 is defined as the drug concentration that reduces parasite growth by 50% compared to the untreated control and is calculated as the inflection point of the sigmoidal function that fits the curve.
		<ol>
			<li>In the statistical analysis software, click on the Analyze button, select Non-linear regression (curve fit) in the xy analysis list, and click Ok.</li>
			<li>In the model tab of the Parameters window, in the dose-response - inhibition group of built-in equations, select the option dose-response method: log(inhibitor) vs. response - Variable Slope (four parameters). Leave all the other tabs at default values; click Ok.</li>
			<li>Click on the results section of the statistical analysis software to find the IC50 value, the SD, and the goodness of the fit.</li>
			<li>Click on the graph section to find the xy graph of the logarithmic concentration of the drug versus the absorbance values. Look for the curve fit is also graphed in a different color. 
			NOTE: A free online IC50 calculation tool can be found at&#160;https://www.aatbio.com/tools/ic50-calculator.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have no conflict of interest to disclose.

This paper describes an assay based on determining the cytoplasmic &#946;-galactosidase activity released due to membrane lysis of T. cruzi epimastigotes, trypomastigotes, or infected cells with amastigotes in the presence of the substrate CPRG. We used T. cruzi Dm28c/pLacZ parasites, a stable parasite strain obtained after transfection with a &#946;-galactosidase-bearing plasmid constructed by Buckner and co-authors10. This assay has been used to search for antitrypanocidal comp...

Chagas disease (ChD), or American trypanosomiasis, is a parasitic disease caused by the flagellated protozoan, Trypanosoma cruzi (T. cruzi). ChD begins with an asymptomatic or oligosymptomatic acute phase that is usually undiagnosed, followed by a lifelong chronic phase. In the chronicity, ~30% of patients manifest-decades after the infection-a variety of debilitating conditions, including myocardiopathy, mega-digestive syndromes, or both, with a mortality rate ranging from 0.2% to 20%1,2,3. Asymptomatic chronic patients may have no clinical signs but remain seropositive throughout their life.
Estimations suggest that ~7 million people are infected worldwide, mostly from Latin America, where ChD is endemic. In these countries, T. cruzi is mainly transmitted through infected blood-sucking triatomine bugs (vector-borne transmission) and less frequently by oral transmission through the ingestion of food contaminated with triatomine feces containing the parasites2. Additionally, the parasite can be transmitted via the placenta from chagasic mothers to newborns, through blood transfusions, or during organ transplantation. These vector-independent ways of acquiring the infection and human migration have contributed to the worldwide spread of the disease, evidenced by an increasing number of cases in North America, Europe, and some African, Eastern Mediterranean, and Western Pacific countries4. ChD is considered a neglected disease as vector-borne transmission is closely associated with poverty and is a leading public health issue, especially in Latin American low-income countries. Although there are available treatments, mortality due to ChD in Latin America is the highest among parasitic diseases, including malaria2.
There are two registered drugs for ChD treatment introduced in the late 1960s and early 1970s: nifurtimox and benznidazole5. Both drugs are effective in the acute phase of the disease in adults, children, and congenitally infected newborns, as well as in children with chronic infection, where cure is usually achieved. However, only a few people are diagnosed early enough to be treated in time. According to the latest clinical trials, both drugs have important limitations in adults and were ineffective in reducing symptoms in people with chronic disease; hence, their use in this stage is controversial. Other drawbacks are the prolonged treatment periods required (60-90 days) and the frequent, severe adverse effects observed, which lead to discontinuation of therapy in a proportion of infected people6,7. It is estimated that fewer than 10% of the people with ChD have been diagnosed, and even fewer have access to treatment, as many affected individuals live in rural areas with no or scarce access to healthcare8. These facts highlight the urgent need to find new drugs against T. cruzi to allow for more efficient, safe, and applicable-to-the-field treatments, especially for the chronic phase. In this regard, another challenge in the development of more efficacious compounds is the limitation of systems for assessing drug efficacy in vitro and in vivo9.
Although chemical biology and genomic approaches for the identification of potential drug targets have been used in kinetoplastid parasites, the available genomic tools in T. cruzi are limited in contrast to T. brucei or Leishmania. Thus, the screening of compounds with trypanocidal activity is still the most used approach in the search for new chemotherapeutic drug candidates against ChD. Usually, drug discovery in T. cruzi must start with testing the effects of a new drug in an in vitro assay against the epimastigote stage. For decades, the only way for measuring the inhibitory effects of candidate compounds on T. cruzi was manual microscopic counting, which is laborious, time-consuming, and operator-dependent. Moreover, this approach is suitable for assaying a small number of compounds but is unacceptable for high-throughput screening of large compound libraries. Nowadays, many investigations begin with the analysis of a vast number of compounds from different origins that are assayed in vitro, testing their capacity for inhibiting parasite growth. Both colorimetric and fluorometric methods have been developed to increase throughput in these assays, improving the objectivity of the screening and making the whole process less tedious9.
One of the most widely used colorimetric methods is based on the &#946;-galactosidase activity of transfected parasites first described by Bucknet and collaborators10. The &#946;-galactosidase enzyme expressed by the recombinant parasites hydrolyzes the chromogenic substrate, chlorophenol red &#946;-D-galactopyranoside (CPRG), to chlorophenol red, which can be easily measured colorimetrically using a microplate spectrophotometer. Thus, parasite growth in the presence of a variety of compounds can be simultaneously evaluated and quantitated in microtiter plates. This method has been applied to test drugs in epimastigote forms (present in the insect vector), trypomastigotes, and intracellular amastigotes, the mammalian stages of the parasite. Further, several recombinant T. cruzi strains transfected with the pBS:CL-Neo-01/BC-X-10 plasmid (pLacZ)10 to express the Escherichia coli &#946;-galactosidase enzyme are already available (and new ones can be constructed), which allows the evaluation of parasites from different discrete typing units (DTUs) that may not behave equally toward the same compounds10,11,12,13. This method has already been successfully used to evaluate compounds for activity against T. cruzi in low- and high-throughput screening12,13. Similar approaches have also been used in other protozoan parasites, including Toxoplasma gondii and Leishmania mexicana14,15.
This paper describes and shows a detailed method for an in vitro drug screening against all life cycle stages of T. cruzi using parasites expressing &#946;-galactosidase. The assays presented here have been performed with a &#946;-galactosidase-expressing T. cruzi line obtained by transfection of T. cruzi Dm28c strain from DTU I13 with pLacZ plasmid (Dm28c/pLacZ). Additionally, the same protocol could be easily adapted to other strains to compare the performance between compounds and between T. cruzi strains or DTUs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 L beaker</td>
			<td>Schott Duran</td>
			<td>10005227</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette sterile</td>
			<td>Jet Biofil</td>
			<td>GSP211010</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipette sterile</td>
			<td>Jet Biofil</td>
			<td>GSP010005</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Corning</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Benznidazole</td>
			<td>Sigma Aldrich</td>
			<td>419656</td>
			<td>N-Benzyl-2-nitro-1H-imidazole-1-acetamide</td>
		</tr>
		<tr>
			<td>Biosafty Cabinet</td>
			<td>Telstar</td>
			<td>Bio II A/P</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube 15 mL conical bottom sterile</td>
			<td>Tarson</td>
			<td>546021</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube 50 mL conical bottom sterile</td>
			<td>Tarson</td>
			<td>546041</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Sanyo</td>
			<td>MCO-15A</td>
			<td></td>
		</tr>
		<tr>
			<td>CPRG</td>
			<td>Roche</td>
			<td>10 884308001</td>
			<td>Chlorophenol Red-&#946;-D-galactopyranoside</td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>Thermo Fisher Cientific</td>
			<td>12100046</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sintorgan</td>
			<td>SIN-061</td>
			<td>Dimethylsulfoxid</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Internegocios SA</td>
			<td>FCS FRA 500</td>
			<td>Sterile and heat-inactivated</td>
		</tr>
		<tr>
			<td>G418 disulphate salt solution</td>
			<td>Roche</td>
			<td>G418-RO</td>
			<td>stock concentration: 50 mg/mL</td>
		</tr>
		<tr>
			<td>Glucose D(+)</td>
			<td>Cicarelli</td>
			<td>716214</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Nalgene</td>
			<td>3663-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Frontier Scientific</td>
			<td>H651-9</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Cicarelli</td>
			<td>867212</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Infusion</td>
			<td>Difco</td>
			<td>226920</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL</td>
			<td>Tarson</td>
			<td>500010-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Spectrophotometer</td>
			<td>Biotek</td>
			<td>Synergy HTX</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Cicarelli</td>
			<td>834214</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Cicarelli</td>
			<td>750214</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Boeco</td>
			<td>BOE 01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P-40</td>
			<td>Antrace</td>
			<td>NIDP40</td>
			<td>2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Statistical Analysis software</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Cicarelli</td>
			<td>929211</td>
			<td>NaHCO3</td>
		</tr>
		<tr>
			<td>Sorvall ST 16 Centrifuge</td>
			<td>Thermo Fisher Cientific</td>
			<td>75004380</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 flasks</td>
			<td>Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptose</td>
			<td>Merck</td>
			<td>1106760500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>ATCC</td>
			<td>CRL-1587</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro drug screening against all life cycle stages of trypanosoma cruzi using parasites expressing &#946;-galactosidase

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi.
T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission.
This paper describes an assay based on measuring the activity of the cytoplasmic &#946;-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red &#946;-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a &#946;-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.

Following the protocol described above, &#946;-galactosidase-expressing Dm28c epimastigotes were incubated with 6 concentrations of BZN (2.5, 5, 10, 20, 40, 80 &#181;M) (or compounds of interest) for 72 h. After this period, CPRG reagent was added along with detergent, which lyses the cells and releases &#946;-galactosidase. CPRG is cleaved by the &#946;-galactosidase to produce chlorophenol red, leading to a change in color from yellow to reddish (Figure 2A). Chlorophenol red was measured b...

Watch this Scientific Journal Video about In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing ÃŽÂ²-galactosidase at JoVE.com

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

We describe a high-throughput colorimetric assay measuring &#946;-galactosidase activity in three life cycle stages of Trypanosoma cruzi, the causative agent of Chagas disease. This assay can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many ...

in-vitro-drug-screening-against-all-life-cycle-stages-trypanosoma

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

3.1K Views.  Universidad Nacional de Rosario (UNR). We describe a high-throughput colorimetric assay measuring β-galactosidase activity in three life cycle stages of Trypanosoma cruzi, the causative agent of Chagas disease. This assay can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner. 

Video: In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a &#34;green&#34; biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme&#39;s protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results.
Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY&#160; II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input.
Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Crystallographic fragment screening at the Helmholtz-Zentrum Berlin is performed using a workflow with dedicated compound libraries, crystal handling tools, fast data collection facilities&#160;and largely automated data analysis. The presented protocol intends to maximize the output of such experiments to provide promising starting points for downstream structure-based ligand design.

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...