During HIV infection, both viruses and microvesicles called exosomes are produced by virus-infected cells. The overall goal of this procedure is to separate exosomes from the virus particles in plasma samples from HIV infected patients. This method can help answer key questions in the exosome field, such as what proteins or any molecules are present in exosomes produced during HIV infection.
Demonstrating the procedure will be Dr.Ming Bo Huang, instructor in the department of microbiology, biochemistry and immunology and a member of the HIV/AIDS group here at Moorehouse School of Medicine. After collecting human blood and isolating the plasma according to the text protocol, add 10 milliliters of 1X PBS to 10 milliliters of plasma in a 15 milliliter tube. Centrifuge the sample at 10, 000 times G and four degrees Celsius for 30 minutes to remove celular debris.
With a sterile serological pipette, transfer the cleared plasma supernatant to a clean, 25 milliliter ultracentrifuge tube. Next, centrifuge the cleared plasma at 200, 000 times G and four degrees Celsius for two hours to separate large vesicles. Then carefully remove the supernatant and discard in the biohazard waste.
Using one milliliter of 1X PBS, resuspend the pellet and incubate at room temperature for 30 minutes, swirling gently to dislodge and separate particles. Add 24 milliliters of PBS to the just resuspended exosome virus solution in a 25 milliliter ultracentrifuge tube and dinphur the tube five times to mix. Then spin again at 200, 000 times G and four degrees Celsius for two hours before discarding the PBS wash solution in the biohazard waste.
To prepare six to 18%velocity gradients of iodixanol, begin with a 60%stock solution of iodixanol reagent and use 1X PBS to dilute it to 6%and 18%Turn on the stirrer and into a dual chamber gradient maker, pipette 5.5 milliliters of the 18%solution into the stirred chamber and 5.5 milliliters of the 6%solution into the reservoir chamber. Then, open the stopcock, turn on the pump and allow each solution to flow into a 14 milliliter ultracentrifuge tube. Next, carefully layer one milliliter of the exosome virus solution onto the top of each 11 milliliter gradient.
Then, with an SW 40 Ti swinging bucket rotor, centrifuge the gradients at 250, 000 times G and four degrees Celsius for two hours. In the meantime, label 12 1.5 milliliter microcentrifuge tubes. After the spin, remove one milliliter from the top of the gradient and transfer to tube number one.
Then transfer the remaining one milliliter fractions to tubes two through 12 in sequential order. Make the assay reagent by mixing 100 parts of 1X PBS, two parts of substrate and five parts of color indicator. Next, transfer 50 microliters in duplicate of each of the 12 one milliliter gradient fractions to the wells of a 96 well microtiter plate.
Prepare a set of standards by first making a 2, 000 milliunits per milliliter ACHE stock solution in PBS. Then, make 11 two-fold serial dilutions of the stock and add 50 microliters of each dilution beginning with 2, 000 milliunits per milliliter to a single well of a 96 well microtiter plate. Add 200 microliters of assay reagent mixture to each well of standards and gradient fractions and incubate in a plate reader for 20 minutes to allow for color development.
Finally, using a fluorescence microplate reader, measure ACHE activity at a wavelength of 450 nanometers. Carry out additional assays according to the text protocol. As shown here, isolated exosomes identified by ACHE activity segregated in lower density fractions one to three at the top of the iodixanol gradients, whereas virus particles identified by HIV antigen P24 segregated in the higher density fractions 10 through 12.
This table shows the analysis of exosomes and unfractionated plasma for 21 cytokines and chemokines using a multiplex assay. As indicated here, all 21 cytokines and chemokines were detected in exosomes from HIV-1 positive individuals and their levels were significantly elevated as compared to plasma and exosomes from HIV-1 zero one negative controls. In this figure, peripheral blood mononuclear cells or PBMCs from uninfected donors were exposed to pooled exosomes from HIV-1 positive or negative individuals.
By 48 hours post-exposure, levels of the activation mark, CD38, on the surface of naive and central memory CD4 positive and CD8 positive t-cells were significantly elevated if they had been exposed to HIV-1 positive exosomes compared to HIV negative treatment. During the acetylcholinesterase assay, it's important to remember to shield the plate from strong light. This technique has paved the way for researchers in the field of HIV/AIDS to explore the role of exosomes in HIV infection and in immune activation.
