The overall goal of the following experiment is to observe the innate immune response to malaria in a chronic HIV infection setting. This is achieved by first culturing live P falses parum parasites in human red blood cells as a second step peripheral blood mononuclear cells obtained from HIV positive and uninfected. Donors are co cultured with the malaria parasite infected red blood cells.
Ultimately flow cytometric analysis is used to assess the innate immune cell cytokine activity of people living with HIV compared to that of uninfected controls. This method can help address key questions in the co-infection field. Like what does the presence of one infection like HIV impact on the immune response of a second infection like malaria To synchronize the parasites, spin down the parasite culture and lyce the trophozoites stage parasites in the pellet with 19 volumes of 37 degrees Celsius alanine solution at room temperature After 15 minutes, spin down the cells in RPMI zero two times.
Adjusting the hematocrit to 3%in R-P-M-I-A after the second wash. Then gas the flask and return it to the 37 degree Celsius incubator until the trophozoites reach a five to 10%parsit to calculate the parsit of the sample. Dispense a 10 microliter sample from the blood layer onto a glass slide and use a second glass slide to create a thin film of the blood after the blood has dried.
Stain the slide using a HEMA three staining kit according to the manufacturer's protocol. To calculate the total number of infected red blood cells. Count 300 cells by light microscopy and compare the number of dark purple stained parasitized erythrocytes to the number of light pink uninfected cells to determine the hematocrit.
Use a hemo cytometer to determine the number of red blood cells per milliliter of parasite culture. Then spin down the parasite culture again and resuspend the cells at six times 10 to the six parasitized erythrocytes per milliliter in R-P-M-I-S plus to isolate the human PBMC dilute blood samples obtained from HIV positive or HIV negative donors in an equal volume of cold DPBS. Next at 15 milliliters of room temperature Fial into a 50 milliliter tube for each.
25 milliliters of blood DPBS solution, followed by careful layering of up to 25 milliliters of the blood and DPBS solution. With a sterile plastic transfer pipette separate the cells by centrifugation and then use a sterile plastic transfer pipette to collect the PBMC from the interface. Transfer up to 20 milliliter aliquots of the PBMC into individual 50 milliliter tubes.
Then bring the total volume of each tube up to 50 milliliters with sterile cold DPBS. Now spin down the cells again and reus suspend the pellet in 50 milliliters of fresh sterile DPBS for two more washes after the final wash. Reus suspend the cells in 10 milliliters of R-P-M-I-S plus for counting and then adjust the PBMC to a final concentration of 10 times 10 to the six cells per milliliter in R-P-M-I-S plus to set up the co-infection.
Cultures begin by seeding one times 10 to the six, the freshly isolated HIV positive and HIV negative PBMC in 100 microliters of R-P-M-I-S plus per well into a 24 well plate. Bring the total volume of each well up to 500 microliters with 400 microliters of R-P-M-I-S plus then in triplicate and in a total volume of 500 microliters per well at three times 10 of the six uninfected red blood cells or three times 10 of the six parasitized red blood cells to the HIV infected and HIV uninfected cells. Place the co-infection plate in a cell culture incubator.
Then after the appropriate experimental time period, pellet the cells and collect 700 microliters of culture supernatant from each. Well centrifuge the supernatant to remove any debris, then aliquot the samples as appropriate. Label the tubes and freeze them at minus 20 degrees celsius or below for later analysis.
Six to eight hours prior to cell specific cytokine production analysis. Treat the samples with one microliter of 1000 D per felden, a per well at the end of the incubation. Place the plate on ice for 15 minutes.
Then use a sterile pipette to remove the cells from the bottom of the plate and transfer them into labeled micro centrifuge tubes. Now spin down the cells and resuspend the pellets in 500 microliters of flow cytometry buffer. Divide the cells up so that each sample has at least one tube for full antibody staining.
One tube for fluorescent minus one control antibody staining also include a single unstained flow cytometry control. Then label the full antibody staining and the fluorescent minus one control cells in 50 microliters of flow cytometry buffer with a pre titrated concentration of fluoro. Four conjugated antibodies to the desired cell surface markers at four degree Celsius and protected from light after 20 minutes.
Wash the samples in one milliliter of flow cytometry buffer and at 100 microliters of cyto fixed cyto perm solution to each tube. After 20 minutes at four degrees Celsius in the dark, wash the cells in one milliliter of permeation wash buffer, spin down the cells and res suspend the pellets in 100 microliters of permeation wash buffer, adding a pre titrated concentration of fluoro four conjugated antibodies to the desired intracellular markers of interest to the full antibodies. Staining samples.
Incubate all of the tubes for 30 minutes at four degrees Celsius, protected from light. Then wash the samples in one milliliter of permeation wash buffer per tube. Finally fix the pellets in 300 microliters of low cytometry buffer, supplemented with 1%paraform aldehyde for 15 minutes to neutralize the HIV before analysis by flow cytometry in these graphs, the levels of interferon gamma production from natural killer T cells in HIV positive and HIV negative individuals exposed to parasitized red blood cells or control uninfected red blood cells are shown.
Note, the suppressed innate immune response to malal infection observed for the HIV positive PBMC as evidenced by their lack of interferon gamma production in the presence of the parasitized red blood cells compared to the robust cytokine production exhibited by PBMC from HIV negative individuals. So don't forget that working with HIV is extremely hazardous and precautions such as performing all steps in a level two biosafety cabinet, wearing the appropriate safety equipment and avoiding the use of glass sharp objects and an aspirator should be done while performing these protocols.
