This assay methodology, it's a major advancement in the knowledge of cell immunity against blood stage malaria, helping to uncover new therapeutic targets and to accelerate the progress of malaria vaccine. The establishment of a plasmodium in vitro assay that can be used for human or animal samples is valuable for a better understand of malaria pathogenesis. Protective malaria immunity is not completely understood.
For the development of new therapies, it is essential to explore mechanisms of cell immunity to the malaria parasitic stage. Demonstrating the procedure will be me, Luna de Lacerda, and Christopher Gomes, an undergrad student from Caroline's lab. To begin, aspirate 100 microliters of heparin solution into a one-milliliter syringe with a 26-gauge needle.
Place the mouse on its side and perpendicularly insert the needle just below the elbow, through the ribs, and into the heart. Pull out the syringe plunger slowly and rotate the needle until 0.5 to one milliliter of blood is obtained. Aseptically clean the left side of the mouse with 70%ethanol.
With surgical scissors, make a cut on the left side of the mouse, passing through the skin and peritoneum. Locate the spleen and remove it. Place the mouse spleen in a Petri dish with five milliliters of complete medium to purify the desired effector cell population.
Place a 100-micromolar cell strainer over a 50-milliliter conical tube and transfer the excised spleen into the cell strainer using anatomical forceps. Mash the spleen through the strainer with a syringe plunger. Wash the cells through the strainer with 10 milliliters of complete media.
Centrifuge the cells at 300 G for 10 minutes at four degrees centigrade. Then, discard the supernatant. Resuspend the cell pellet in two milliliters of cold RBC lysis buffer and incubate the suspension for five minutes on ice.
Wash the cell suspension with 10 milliliters of four-degrees-centigrade complete media and remove any cell clots between washes for spleens from plasmodium-infected mice. Centrifuge the cells at 400 G for five minutes at four degrees centigrade. Then, discard the supernatant.
Now place an LS or LD column in the magnetic field and prepare the column by rinsing it with three milliliters of MACS buffer. Next, apply cell suspension onto the column. After washing the column three times with three milliliters of MACS buffer, collect flow-through containing the splenic cells.
Harvest 10 microliters of the splenic cells and add 10 microliters of trypan blue to check cell viability. Add the cells to the hemocytometer. Count the splenocytes in a trypan blue solution and check cell viability.
Centrifuge all splenocytes and discard the supernatant. Resuspend the cell pellet in 40 microliters of MACS buffer. Add 10 microliters of a biotin antibody cocktail to the cells, mix well, and incubate for five minutes on ice.
After incubating the anti-biotin microbeads on ice for 10 minutes, place the MACS LS column in the magnetic field support and prepare the column by rinsing it with three milliliters of MACS buffer. Apply cell suspension onto the column. Wash the column three times with three milliliters of MACS buffer and collect the flow-through containing all unlabeled cytotoxic cells.
After centrifuging the collected blood at 350 times G for five minutes, discard the serum and resuspend the blood in one milliliter of RPMI without FBS. After placing the LS column in the magnetic field support, rinse with RPMI and pass the RBC suspension through the column. Once the cells pass through the column, centrifuge the flow-through, discard the supernatant, and reapply one milliliter of the flow-through into the column to isolate more iRBCs.
Wash the column twice with five milliliters of RPMI. Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty. Add five milliliters of RPMI, remove the column, and purge the iRBCs into a new 15-milliliter tube.
Dilute one microliter of a 10-millimolar CFSE solution in one milliliter RPMI without FBS. After resuspending the RBCs in 500 milliliters of RPMI without FBS, add 500 milliliters of the diluted CFSE and incubate for eight minutes at room temperature, protected from light. Wash the cells thrice with 14 milliliters of complete medium and centrifuge the cells twice.
For cytotoxic lymphocyte co-culture, plate the cells in a 96-well round-bottom plate. Add the purified lymphocytes and CFSE-labeled iRBCs in the desired effector/target cell ratio to a final volume of 200 milliliters and homogenize. Prepare each condition in triplicate and incubate the cells at 37 degrees centigrade.
The iRBCs were purified from P.yoelii-infected mice using LS columns. The purification is highlighted by blood smears from blood collected before and after column enrichment. The gating strategy needed to assess the percentage of RBC lysis is presented here.
The stopping gate is set into the counting beads population. The gating strategy is started by selecting the single cells, excluding debris, based on the forward scatter peak height-to-area ratio, followed by the selection of the RBC population in Ter119+and CD8-Then, select live IBCs as CFSE high positive. Here is the analysis applied to an experimental example using different effectors to target ratios, displaying live RBCs after the co-culture.
A graphical representation of the RBC lysis percentage is shown, calculated based on this formula. The significance between the groups using cytotoxic CD8 or naive CD8 was assessed by two-way ANOVA with multiple comparisons. The current method explores RBC lysis by direct cell contact with killer cells, and it can be planned to uncover the involved mechanisms using blocking antibodies to specific receptors or molecules.
This in vitro kill assay presents a novel strategy to clarify the mechanisms of cell-mediated immunity to blood stage malaria, which will help the progress of new malarial therapies and vaccines.
