The overall goal of this procedure is to select plasmodium, fpar, and parasites for binding to human brain endothelial cells. This is accomplished by first incubating a culture of plasmodium, fpar, and parasites, which contain a mixture of infected and uninfected red blood cells with a layer of human brain endothelial cells, the next step is to wash off uninfected and unbound red blood cells by washing several times with culture. Medium, fresh red blood cells are then added to allow further growth of the parasite population.
The final step a few weeks later is to repeat the overall selection as soon as a sufficient paras is reached. Ultimately, microscopy is used to show a high amount of parasites bound to HBE five i. This method can help answer key questions in the cerebral malaria field, such as the identification of parasite ligands or host receptor molecules involved in the cyto endurance process.
Prepare human red blood cells from group O positive whole blood by separating the red blood cells from the white blood cells by passage through a leukocyte depletion filter. Centrifuge the cells at 1000 G for 10 minutes and Reese's spend the pellet with all PMI. Incomplete, medium.
Repeat this wash step once more, then re suspend the pellet. In our PMI incomplete medium at 50%hematocrit, red blood cells are stored at four degrees Celsius for a week. Culture Odium Valspar infected red blood cells.
With RPMI complete medium at 2%hematocrit and incubate at 37 degrees Celsius with 3%carbon dioxide, 1%oxygen, and 96%nitrogen every day. Make a gem smear to assess the developmental stage of the parasites. Change the medium daily by spinning the culture at 500 G for five minutes.
Aspirating out the supernatant and adding fresh RPMI complete medium about once a week. Synchronize the parasites by treatment with aqueous 5%D sorbitol. The selection assay may be performed the day after a ring stage culture with a minimum of 5%to 10%Parsit is observed.
In addition. Enough parasite culture to form a 30 microliter P cell volume is needed per 60 millimeter dish of hbe five eye cells. Prepare a 60 millimeter tissue culture dish to receive the human brain microvascular endothelial cell line or H back fivei by adding two micrograms per centimeter squared of fibronectin and sterile saline to the dish.
Incubate the dish at 37 degrees Celsius for five to 20 minutes, then aspirate the fibronectin solution. Then harvest the HBE five I cells from a 25 centimeter square tissue flask by ionization and centrifugation Reese, suspend the cell pellet in 10 milliliters of DMEM, complete medium. Then add 1.5 milliliters of this cell suspension to the fibronectin treated tissue culture dish and incubate at 37 degrees Celsius and 5%carbon dioxide for two to three days until the culture reaches 50 to 100%co fluency and the parasite cultures are ready for cyto aian on the day of the assay.
The HBE five I culture should be at 50 to 100%co fluency. While the parasite culture should be at the pigmented trophozoites stage with at least five to 10%paras and 2%hematocrit. First pipette 1.5 milliliters of parasite culture from the culture flask and transfer to a 15 milliliter centrifuge tube.
Centrifuge the culture at 500 G for five minutes and re bend in 10 milliliters. A freshly made warm DMEM incomplete, medium. Repeat this wash step a second time after the final centrifugation is complete.
Asberry the DMEM incomplete, medium, and re spend the 30 microliter pellet in 1.5 milliliters of DMEM. Incomplete medium, supplemented with 1%BSA. Next, retrieve the 60 millimeter dish of hvac.Five.
Eye cells aspirate the culture media and wash twice with three milliliters of DMEM. Incomplete medium. Add the solution of parasites, the dish of HPE five eye cells and incubate in the tissue culture incubator for 75 minutes in total.
Twice during the incubation resus, suspend the parasites by gently rocking and swirling the dish following the incubation. Wash the dish five times by aspirating the medium using a plastic pasta pipette to add three milliliters of warm DMEM incomplete medium, and then gently rocking the plate after the final wash, examine the dish under an inverted microscope. If many uninfected red blood cells are still visible, repeat the wash procedure as described above to remove them.
After aspirating the media from the dish Resus suspend 40 microliters packed cell volume of fresh red blood cells In warmed RPMI complete medium and add the cell suspension to the dish. Place the dish in an air sight incubating chamber. Then gas the dish with 3%carbon dioxide, 1%oxygen and 96%nitrogen for three minutes.
And place the chamber in an incubator at 37 degrees Celsius overnight. During this incubation, schiz on stage parasites burst and maites reinve the red blood cells. The next day, harvest the ring stage parasites by washing.
With RPMI incomplete medium in a similar manner as before, but with more vigorous pipetting of media and rocking of the plate. Keep all of the media used, which will contain resuspended red blood cells in a 15 milliliter conical tube. Check the plate under an inverted microscope to ensure that all red blood cells have been removed from the dish centrifuge.
The tube containing the media from the washed plate to pellet the parasites discard the supernatant Resus suspending five milliliters. RPMI complete medium and place the mixture in a 25 centimeter square flask for culturing. The parasites are cultured for two to four weeks until enough material is obtained and a new round of selection can be performed.
Unselected HB three parasites show a low level of binding to hbe five eye cells. This image shows hbe five eye cells fixed with 2%glutaraldehyde and stained with giemsa visualized under the microscope at 1000 times magnification peaches were taken after uninfected red blood cells and unbound infected red blood cells were washed away. After each round of selection, more and more parasites bind into the endothelial cells and the selections can be repeated at shorter time intervals after five rounds of selection.
High binding parasite populations are obtained as shown here with HB three HBE parasites. This table shows a summary of the plasmodium alspar strains that were successfully selected for binding to HE five. I note that HB three was also selected on TNF activated HE five I after five rounds of selection.
The DD two strain showed no increase in binding to HBE five I compared to unselected DD two. This 400 times magnification image shows binding of plasmodium alspar HB three parasites to HD MEK dermal endothelial cells before selection. Here, the binding of plasmodium alspar HB three parasites to the same dermal endothelial cell line is shown after four rounds of selection.
This figure shows binding of plasmodium spar HB three parasites to HP ME pulmonary endothelial cells before selection. Now the binding of plasmodium spar HB three parasites to the H HP ME cells is increased after four rounds of selection. Although the culture medium for hd e, C and H HP EC slightly differs, the protocol used for the selection was identical as with hbe five I.Following this procedure of the methods like R-T-P-C-R or micro analysis can be performed to answer additional questions like characterizing the transcription of variant surface antigen in unselected and selected parasites.
