The overall goal of the experimental technology demonstrated here is to screen compounds against growth and proliferation of intracellular ama goat stage of Lish mania. Don Avani the cause of agent for visceral leishmaniasis with a primary objective to discover new drugs for treatment of lemanis First THP one Human acute monocytic leukemia cells are differentiated in vitro into a adherent macrophages, the differentiated THP one cells and then infected with ProMaster goats of the leash Manaya Donovan parasites, which allows invasion of the ProMaster goats into the TP one macrophages, their transformation into AMAs, goats and intracellular proliferation. Next, the leishmania infected THP one macrophages are exposed to test compounds and standard drugs and then undergo controlled lysis, rescue of a master goats transformation of residual, a master goats to ProMaster goats and quantification of ProMaster goats by colormetric cell growth assays.
Results obtained that show the anti mania effect of test compounds and standard drugs on intracellular growth and proliferation based on quantification of intracellular am master goats. Lemanis is a major global health threat, especially in tropical areas of the world. New drugs with better therapeutic profiles to treat this disease are urgently needed.
We at the National Center for Natural Products Research have launched a multi-directional program for the discovery of new treatments for tropical parasitic diseases. Unlike the Rogen and microscopic image analysis assays, which may not differentiate between the live and dead msst goods, this SA measures only the live intracellular MST goods. We have optimized a parasite rescue and transformation assay with differentiated TP one cells infected in vitro with the liman for screening the pure compound and natural product extract and determination of efficacy against the intracellular limania.
The experimental details of this essay will be demonstrated by Mr.Ra, who is the research and development biologist in my lab and performs this essay and Mr.RA Sahu, who is also working as a research and development biologist in my lab. There are four basic steps in the assay. Step one is seeding and terminal differentiation of THP one cells.
Step two is in vitro infection of differentiated TP one cells with L mania proma goats. Step three is treatment of Lish mania infected macrophages with the test compounds or standard drugs. And step four is quantification of the growth and proliferation of lush mania parasite goats with microscopic image analysis or parasite rescue and transformation assays.
Harvest a four day old culture of THP one macrophages and prepare a cell suspension in our PMI supplemented with 10%heating activated FPS with a concentration of 2.5 times 10 to the five cells per milliliter. Add 25 nanograms per milliliter of PMA to the cells to trigger differentiation. Then transfer 200 microliters of cell suspension to each well of a 96 well plate and each chamber of a 16 chamber glass slide once the cells have been plated, place the slide and the 96 well plate in a 37 degree Celsius incubator and incubate the cells overnight to allow nearly complete cellular differentiation to occur.
For the infection. Use a five to six day old ProMaster goat parasite culture in which most organisms are in the infective cynic stage. Unlike the pro cyclic stage ProMaster goats, which are short ellipsoid cells with a short gel, weekly motil and often found in rosettes, the infective meta cyclic organisms can be recognized by their long cylindrical shape and high motility.
Harvest the parasites and prepare a ProMaster goat suspension of 2.5 times 10 to the six parasites per milliliter in 2%F-B-S-R-P-M-I retrieve the differentiated THP one cell cultures from the incubator processing the 96 well plate and glass slide. In parallel, remove the supinate from the cultures, then wash the cells by adding pre-war serum free medium. Remove the serum free medium and replace it with 200 microliters per well of ProMaster goat suspension, achieving a ratio of 10 parasites for each THP one cell.
For control wells, add 200 microliters of 2%F-P-S-R-P-M-I medium instead of the parasite suspension. After adding the parasites, incubate the co cultures at 37 degrees Celsius for 24 hours to allow the infection to occur from stock solutions of the drug to be tested. Prepare six serial one to five dilution in 2%F-B-S-R-P-M-I, such the concentrations of each dilution is twice the intended final test.
Concentration as controls standard anti manial drugs such as amphotericin b pentamidine, melter, Fosse, and sodium stib gluconate may be used before adding the drugs to the co cultures. Repeatedly wash the TP one cells in the plate and slide as described previously. Wash the cells at least five times to remove any non internalized ProMaster goats.
Then add 100 microliters of 2%F-B-S-R-P-M-I and 100 microliters of each drug solution to each well or chamber, and incubate the plate and slide at 37 degrees Celsius for 48 hours following the drug treatment. Wash the chamber. Slide three times in serum free medium.
Peel off the plastic chambers from the slide and fix the cells by immersing the slide in methanol for 30 seconds. Place the slide in a biohazard hood with lamina flow to allow it to dry. In the meantime, prepare a five x solution of cyber green.
Once the slide is dry, immerse it in cyber green to stain the cellular and parasitic DNA. Incubate the sample in the dark for 15 minutes of room temperature. After the staining step, wash the slide with water and let it dry.
Using mounting medium, place a glass cover slip over the slide. Then observe the samples using a fluorescent microscope and capture images of each chamber in uninfected cells only the macrophage nucleus is visible in infected cells. The parasite nuclei and canlas can be seen in the cellular cytoplasm attesting to the presence of intracellular l Donny parasites.
To quantify the infection, use image J to calculate the percent infectivity, which is the number of am master goat nuclei times 100 over the number of macrophage nuclei for the parasite rescue assay. First wash the 96 well plate co-culture three times with serum free RPMI medium to liso cells at 20 microliters of 0.05%S-D-S-R-P-M-I. To each well shake the plate for 30 seconds.
Then add 180 microliters of 10%F-B-S-R-P-M-I per well. The time for SDS treatment should not exceed 30 seconds to obtain controlled lysis of the macrophage without any effect on viability of rescued a master. Goats incubate the plate at 26 degrees Celsius for 48 hours to allow transformation and proliferation of the rescued visible a master into pro master goats.
To quantify the pro master goats, add 10 microliters of alamar blue to each well of the 96 well plate and incubate it at 26 degrees Celsius overnight. Following this incubation, quantify the live parasites by reading the plate in a standard fluorescence fluorimeter. In the absence of drugs, the percentage infection increases with the number of a master goats present relative to the number of macrophages.
In addition, the quantity of surviving parasites determined by rescue assay correlates well with the percentage infection in the presence of increasing concentrations of amphotericin. B.The number of rescued parasites decreases in a dose dependent manner. The parasite rescue and transformation assay may be summarized with sequential microscopic views.
This picture shows intact THP one cells in culture infected with lush mania Amma goats. These infected THP one cells are subjected to controlled lysis to rescue live amma goats. The live Amma goats rescued from the infected THP one cells transforming culture into ProMaster goats.
These transformed ProMaster goats grow and proliferating culture. The overall number of ProMaster goats in culture is directly proportional to the number of live intracellular oma goats. This essay has been optimized for the control license of limania infected THV one cells.
The objective was to optimize the conditions for detergent treatment, which will almost complete lysis of THV one cells with minimal effect on the TY of the rescue MST codes. This step is very critical in this assay. The parasite rescue assay demonstrator here can also be applied for aerating infectivity of different species of linia parasites causing cutaneous or mu cutaneous leishman acid in different micro heart cell lines, and also different lenia field as isolates or genetically manipulated parasite cell lines.
Both parasite rescue as SA and image analysis as SA show comparable results. They have the potential for automation and application for large scale screening. We have successfully applied this essay for screening more than 16, 000 fractions prepared by high throughput fractionation of natural product accepts available at our center.
We have also optimized the image analysis as essay by combining the images captured under DIC and fluorescein filters and and quantification of the infection with IM image j. Image J is a publicly available Java based image processing program originally developed at the NIH. Through this assay, several natural product fractions with novel compounds have been identified as new leads for inte mal tract discovery.
