An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes
Transcrição
Adjust the purified infected erythrocyte suspension by 3.3 times 10 to the 7th cells per milliliter in parasite culture medium containing ethidium bromide to a final concentration of 2.5 micrograms per milliliter. Next, remove the blocking solution from one 96-well plate by flicking the plate into a waste container. Then, remove any excess over a paper towel, and add 30 microliters of the ethidium bromide label infected erythrocyte suspension to all but one well in the upper half of the plate. Add 30 microliters of parasite culture medium to the empty well, and incubate the plate for 10 minutes at room temperature, protected from light.
At the end of the incubation, add 170 microliters of parasite culture medium to each well, and sediment the infected erythrocytes at the bottom of the plate by centrifugation. Remove the supernatant by flicking the plate into a waste container, and wash the ethidium bromide-labeled infected erythrocytes two more times with 200 microliters of parasite culture medium per well, as just demonstrated.
After the second wash, use a multichannel pipette to carefully remove the entire volume of supernatant from each well, without disturbing the pellets, and resuspend the ethidium bromide-labeled infected erythrocytes in 30 microliters of antibody solution, including the appropriate controls and test samples previously prepared in parasite culture medium. Then, place the plate at 37 degrees Celsius for 45 minutes, protected from light.
While the infected erythrocytes are being opsonized, collect the THP-1 cells by centrifugation, and resuspend the pellet in 12 milliliters of fresh pre-warmed THP-1 cell culture medium.
After a second centrifugation, resuspend the pellet in 1 milliliter of THP-1 cell culture medium for counting, and adjust the cell concentration to 5 times 10 to the 5th cells per milliliter in THP-1 cell culture medium. Remove the blocking solution from the second 96-well plate, and add 100 microliters of THP-1 cells to each well. Then, place the plate in the cell culture incubator.
At the end of the opsonization incubation, add 170 microliters of parasite culture medium to each well of the opsonization plate, and sediment the infected erythrocytes to the bottom of the plate by centrifugation. Wash the opsonized, infected erythrocytes two times with 200 microliters of parasite culture medium per well using a multichannel pipette to carefully remove the supernatant after the second wash.
Resuspend the opsonized, infected erythrocytes in 100 microliters of pre-warmed THP-1 cell culture medium per well, and transfer 50 microliters of each opsonized, infected erythrocyte suspension to a corresponding well in the phagocytosis plate. When all of the cells have been added, place the plate in the cell culture incubator for no more than 40 minutes, protected from light.
At the end of the incubation, stop the phagocytosis by centrifugation at 4 degrees Celsius. Remove the supernatant, and resuspend the pellets with 150 microliters of room-temperature ammonium chloride lysing solution per well. After exactly three minutes, add 100 microliters of ice-cold 2% FBS in PBS to stop the lysis, and sediment the intact cells by centrifugation.
Next, wash the cells three times with 200 microliters of fresh ice-cold 2% FBS in PBS, per well, per wash. After the final wash, resuspend the cell pellet in 200 microliters of fresh ice-cold 2% FBS in PBS per well.
For flow cytometry acquisition and analysis, immediately load the cells onto a flow cytometer, and use the linear forward versus linear side-scatter plot for the wells without infected erythrocytes to gate the THP-1 cells. Acquire 10,000 events in this gate, and use the THP-1 cells with the infected erythrocytes opsonized with a positive control to set up a histogram plot to measure the ethidium bromide fluorescence intensity.
For analysis, open the linear forward versus linear side-scatter plot for the wells without infected erythrocytes, and gate the THP-1 cells. Then, set up a positive gate in an FL3 histogram, and copy these gates onto all of the other sample wells to determine the percentage of ethidium bromide-positive THP-1 cells.
