The overall goal of this procedure is to develop an in vitro model to study the effects of plasmodium fpar infected erythrocytes on the replication of HIV one in human primary monocyte derived macrophages. This is accomplished by first isolating plasmodium, false para infected red blood cells using the veac system, and then exposing human monocyte derived macrophages to the infected cells. Next, the red blood cell exposed monocyte derived macrophages are infected with fully replicative HIV or single replication luciferase encoding, HIV.
Finally, the culture supernatants are harvested at days 3, 6, 9, and 12 post-infection for the fully replicative virus for the luciferase encoding virus. The infected cells are lies after 72 hours of infection. Ultimately, viral replication can be monitored by quantifying the amount of HIV one P 24 capsid protein in supernatants by Eliza and viral transcription can be monitored by luciferase quantitation of the cell lysates.
This method can help answer key questions in the field of malaria, HIV co-infections. For example, how does infection with the plasmodium parasite influences HIV replication? Though this method was developed to study interactions between plasmodium and HIV one in macrophages, it could also be applied to other cell types.
For example, dendritic cells, monocytes, or CD four T cells demonstrating the procedure will be Guadalupe Andreani. A postdoctoral fellow in the lab Begin by harvesting red blood cells from the culture plate, wash the recovered cells in 10 milliliters of monocyte derived macrophage medium, and then resuspend the pellet in 12 milliliters of monocyte derived macrophage culture Medium. Now slowly add the cell suspension to a Milton ECS column and let the suspension flow through after washing the column once with fresh medium, remove it from the magnet and flush 12 milliliters of monocyte derived macrophage culture.
Medium through the column, eluding the infected red blood cells into a sterile tube to expose monocyte derived macrophages to infected or uninfected red blood cells. Add the appropriate red blood cell suspension volume to each well of a previously prepared 24 well plate containing monocyte derived macrophages. Incubate the co cultures at 37 degrees Celsius in 5%carbon dioxide after four hours, remove the medium containing the red blood cells and then gently add 600 microliters of endotoxin free PBS to each, well three times aspirating the PBS immediately after each wash.
Now at 200 microliters of ice, cold, sterile water to each well to ly the red blood cells aspirating the water after 20 seconds. Then at 600 microliters of monocyte derived macrophage culture medium to each well and incubate the cultures for 24 hours at 37 degrees Celsius and 5%carbon dioxide. To assess the effect of plasmodium false parem on HIV one replication in monocyte derived macrophages at 300 microliters of monocyte derived macrophage medium containing P 24 of NL four three BO IV one viruses into the appropriate wells similar to the protocol depicted in this figure.
After incubating the co cultures for two hours, wash the cells three times with 600 microliters of endotoxin free PBS. Then at 600 microliters a fresh monocyte derived macrophage medium to each well and continue to incubate the co cultures on days 3, 6, 9, and 12 After the initial viral infection, harvest 200 microliters of each snat replacing the recovered cell suspension with 200 microliters of fresh monocyte derived macrophage.Medium. Thereafter store the harvested supernatants at minus 20 degrees Celsius for later analysis by Eliza.
In order to determine the parasites impact on HIV one gene expression in monocyte derived macrophages at 300 microliters of monocyte derived macrophage medium containing P 24 of the luciferase ENC coated virus of interest to the corresponding wells similar to the protocols illustrated here. After incubating and washing the code cultures as just shown for non luciferase ENC coated virus, remove the medium 72 hours following infection and add 200 microliters of the luciferase assay. Hit lysis buffer now lys the cells at room temperature with gentle shaking and then store the co cultures at minus 20 degrees Celsius.
After thawing the plate transfer 40 microliters of lysate from each well to the corresponding well in a 96 well luminometer plate. Then add 100 microliters of the luciferase assay kit luciferase substrate to each. Well finally measure the firefly luciferase activity in a luminometer.
According to the manufacturer's instructions in this experiment, monocyte derived macrophages were exposed to infected or uninfected red blood cells and then infected with NL four three blen. As just demonstrated the production of the viral protein P 24 was then monitored by ELIZA for HIV one P 24 in cell-free supernatants at different time points following the initial viral infection. Note the significant decrease in the release of viral particles on day 12 as measured by HIV one P 24 capsid protein in the supernat of monocyte derived macrophages pretreated with parasites.
In this experiment, monocyte derived macrophages were exposed to infected and uninfected red blood cells and then infected with luciferase encoded virus as just demonstrated Luciferase expression was then evaluated in cell lysate 72 hours following the initial virus in infection. Monocyte derived macrophage infection with such viruses harboring either exogenous VS.VG or HIV one glycoproteins led to significantly less luciferase production in cells exposed to p false spar. It is noteworthy that VSVG pseudo typed viruses yielded much greater luciferase activity than their JRFL on counterparts due to the greater infection efficiency of VSPG pseudo typed particles.
This technique will allow researchers in the field of malaria, HIV co-infections to explore the interactions between these two pathogens in an in vitro model that is adaptable to the study of several immune cell types.
