The overall goal of this procedure is to generate pure plasmodium falciparum maites in high yield for quantification of their opsonization by human antibodies. This is accomplished by first inhibiting S schon rupture with E 64 to generate membrane enclosed maite structures. In the second step, the free maites are harvested and then stained with AUM bromide for quantification in the final step, the maites are incubated with various human plasma samples and THP one human monocytic cells.
Ultimately, the opsonization of the maites by the different human plasma samples can be differentiated by flow cytometry. The advantages of this technique over existing assays of functional immunity to malaria meite is that intact mes are used and phagocytosis responses are robust and quantifiable. This method can help answer key questions in the malaria immunology field, such as what is the importance of oxidizing antibodies and phagocytosis for immunity against malaria in vaccine or naturally exposed individuals?
Visualization of this technique is critical as the timing of E 64 is essential to ensure the formation of mezo. And because mezo preparation and enumeration is technically demanding After magnetic separation of late stage P falciparum Trophozoites assess parasite maturation by fixing a thin smear of parasites in 100%methanol for 10 seconds and staining them in Giza for three minutes. Examine the stain slide under the microscope to ensure that the parasites almost fill the red blood cells and appear to be in the early zen stage.
If the parasites have not reached the appropriate maturation stage, continue to incubate them until suitably developed. Once suitably mature, treat the isolated schon with 10 micromolar E 64 and incubate them for another six to 10 hours. Following the incubation with E 64, prepare a smear of the treated parasites.
Fix the slide in 100%methanol and stain it with Giza to confirm that at least 50%of the parasites have formed membrane. Enclosed maites pellet the remaining E 64 treated schon for eight minutes at 1900 Gs at room temperature. Then wash the parasites in 50 milliliters of room temperature.
Wash medium to remove any remaining human serum. After discarding the supernatant, resuspend the parasite pellet in two milliliters of wash medium, and then filter the cell suspension through a 1.2 micrometer syringe filter, collecting the maites and hemon crystals in a 10 milliliter tube. Next, attach a small magnetic column to a magnet and equilibrate it with 500 microliters of THP one media.
Pass the filtrate over the magnetic column three times collecting the maites in the flow through each time. Then rinse the column with two milliliters of room temperature. THP one media to collect any remaining maites.
Removal of the hemin is a critical step for purifying the maites. If the maites are not separated from the hemin, then maite hemin clusters will form resulting in inaccurate maite counting by flow cytometry. THP one cells con phagocytose hem hemin, so the meite hemin clusters can also be ceto.
Thus, if the hemin is not removed, the opsonizing potential of the plasma being tested will be overestimated as well. Stain the collected maites in 10 micrograms per milliliter of ahy bromide at room temperature protected from light. After 30 minutes, spin down the maites, discard the supernatant into an appropriate waste container, and wash the maite pellet in four milliliters of THP one media pellet maites.
Then resus suspend the cells in 15 milliliters of TP one media protected from light. To quantify the maites by flow cytometry. Count maites at one in 100, one in 50, and one in 25 dilutions.
Firstly, dispense 940, 930 and 910 microliters of PBS plus BSA into three different fax tubes and add 10, 20, and 40 microliters of purified maites to each tube respectively. Next vortex room temperature counting beads for 30 seconds and then add 50 microliters of the beads to each fax tube containing the diluted maites. Run the three diluted maite samples on a flow cytometer equipped with a 488 nanometer laser, setting a stringent gate for the maites based on the atherium bromide and GFP dual fluorescence gate the beads in a separate channel and acquire events until 2000 beads have been collected.
To quantify the number of maites at each dilution average the three maite concentration measurements. Then resus suspend the maites at eight times 10 to the six maites per milliliter in THP one media to ensure a final ratio of four maites per THP one cell to measure the phagocytosis of the maites. First pellet enough THP one cells for the assay and resus suspend them at 6.7 times 10 to the fifth cells per milliliter in THP one media.
Next at 150 microliters of THP one cells per well in triplicate for each condition to be tested to an FCS blocked 96 well U bottom plate to a final 10 to the fifth cells per well concentration. Place the plate in the incubator until it's time to add the maites. Then add 150 microliters of the isolated maites at eight times 10 to the sixth milliliters per well concentration into a separate FCS blocked 96 well U bottom plate at one well per condition to be tested.
Add 10 microliters of prepared diluted plasma samples to each well of the maites mixing thoroughly to ensure a homogenous solution. Then add 50 microliters of the maite plasma solution to each well of the THP one plate in triplicate for each plasma sample being tested. Mix the samples well and then incubate the co cultures covered in a humidified incubator at 37 degrees Celsius in 5%carbon dioxide.
After 40 minutes, spin down the samples in a pre chilled centrifuge to arrest the phagocytosis. Then wash the cells twice in ice cold fax buffer after the second wash. Fix the cells in 90 microliters of fax fixative and leave them on ice.
Protected from light until acquisition. The maturation stage of parasites prior to E 64 treatment is critical for generating membrane enclosed maites. The parasites should be large, almost fill the red blood cell and show dappled gems staining.
Adding E 64 at this stage will yield membrane enclosed maites after incubation for six to 10 hours. If E 64 is added earlier to trophozoites stage parasites membrane enclosed maites will not be generated. If E 64 is added later to schon schon rupture is uninhibited.
A high level of parasite synchrony is required. Otherwise, a range of all three outcomes described will be seen after E 64 treatment phagocytosis measurement by aum bromide fluorescence enables superior resolution of the parasite phagocytosis over GFP fluorescence alone. Increasing the ratio of maites to TP one cells results in an increased adherence of the maites to the THP one cells.
In the absence of maite specific antibodies, increasing the MEITE to THP one cell ratio also results in an increase in phagocytosis between zero and 78%Opsonization of the maites has been observed using Papua New Guinea plasma samples at a four to one MAITE to THP one cell ratio. These data show examples of the four quartiles of PHA acidic responses from four representative Papua New Guinea individuals. Opsonizing antibodies measured using this assay have been shown to associate with clinical immunity to malaria While attempting this procedure.
The key points to remember to ensure successful PE cytosis are to have highly synchronized parasites to obtain fully matured and intact meite, and to maintain t HB one cell cultures appropriately. The technique for meite purification outlined in this procedure can be adapted to any application requiring high quality malaria maites, such as marizone, invasion, assays, proteomics, or serology. After watching this video, you should have a, a good understanding of how to use the E 64 method to purify maites and to investigate opsonizing antibodies by measuring phagocytosis by THP one cells.
