TITLE: Affinity purification of ookinetes in Petri dishes
AUTHORS: Benito Recio Totoro

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The ookinetes, as they come from the ookinete culture, are suspended in a solution that also contains blood cells and other parasite stages. Often, it is necessary to purify the ookinetes from these other cell types. This is achieved exploiting biological, chemical, and physical properties of the ookinetes that differ from the other cell types present in the ookinete culture.</div><div class = "text-block"><span>This protocol exploits the adhesive and invading characteristics of ookinetes by allowing them to attach to a surface coated with Extracellular Matrix gel (ECM gel). The rest of the cells present in the ookinete culture do not have adhesion properties and can be simply washed away. ECM gel is composed of approximately 1800 proteins, the most abundant are type IV collagen, laminin, entactin, and other proteoglycans. The </span><span style = "font-style:italic;">Plasmodium</span><span> ookinete has several surface proteins that bind to the ECM gel components, such as the P25, and P21/28 protein families that bind to laminin and collagen, and the CTRP, SOAP, and WARP surface or secreted proteins that bind to laminin, and other glycosaminoglycans.</span></div><div class = "text-block">The purification in Petri dishes allows the obtention of a higher amount of ookinetes with the expense of being less pure than the purifications on coverslips. The ookinetes can be re-suspended by the use of dispase II, a protease that cleaves type IV collagen and repurposed for experiments that require them in suspension or to perform organelle and macromolecule extracts.</div></div>

[STEPS]
?. [Ookinete purification]
Thaw the ECM gel overnight at   in ice
4 °C
Keep the ECM gel in ice at all times. ECM gel starts to form a gel at 20°C
?. [Ookinete purification]
Dilute the ECM gel 1:2 or 1:3 with cold ECM buffer, mix well and coat the Petri dishes with .Allow gel formation at  for .
0.5 mL
37 °C
Up to 1:9 ECM gel dilutions can be made with the expense of reducing the ookinete yield.Optionally, the coated Petri dishes can be left at 37°C until the ECM gel is dry, after which the coverslips can be stored at 4°C. Before use re-hydrate the ECM gel by adding ookinete medium for 30 min at 37°C.
?. [Ookinete purification]
Add between  to  of ookinete culture to the Petri dishes and incubate for  at .
2.5 mL
6 mL
20 °C
During incubation, the dishes can be gently shaken to increase the likelihood of contact between the ookinetes and the substrate.
?. [Ookinete purification]
15 min before the incubation ends, tilt the Petri dishes at a 10-15° angle. This will cause the sedimented cells to slide to one side of the container leaving most of the dishes already free of contaminant cells.
?. [Ookinete purification]
Remove the contaminant cells from one side of the dish and add  of PBS from the other side of the dish to create a one-way flow of liquid.Repeat the washes 6 or 8 times.Add ookinete or oocyst medium for further cultivation.
2 mL
?. [Ookinete resuspension]
Alternatively, the ookinetes can be brought back to suspension by digesting the ECM gel with dispase II.After purification, instead of adding culture medium, add  of dispase II and incubate for  at .
2 mL
20 °C
?. [Ookinete resuspension]
Add EDTA at a final concentration of  and mix well.
?. [Ookinete resuspension]
Tilt the Petri dish and transfer the ookinetes into a 15 ml conical-bottom centrifuge tube in ice.
?. [Ookinete resuspension]
Add   of ice-cold PBS and pipette a couple of times to detach the remaining ookinetes.Repeat this process 4 times.Collect the ookinetes and the washings in the same tube.
2 mL
?. [Ookinete resuspension]
, remove the supernatant and repeat.
Centrifuge: 720 1324, 5 min, 4 10
?. [Ookinete resuspension]
Re-suspend the pellet in  ookinete or oocyst medium and count the number of ookinetes in a Neubauer chamber.
1 mL
?. [Mosquito infections]
This method can be used to infect mosquitoes with purified ookinetes. However, the number of ookinetes needs to be increased in order to obtain equivalent infection rates compared to mosquito infections with non-purified ookinete cultures.
This result represents an improvement of what was observed by Munderloh U.G., et al., (1987) with ookinetes purified by Percoll gradients. While Munderloh U.G., et al., (1987) estimated that 110 ookinetes per µl were needed to obtain each oocyst in feedings with enriched ookinete cultures, and 510 ookinetes per µl to obtain each oocyst in feedings with Percoll-purified ookinete cultures, representing a 5-fold reduction in infectivity, we obtained an estimate of 43 ookinetes per µl per oocyst formed in feedings with non-purified ookinete cultures, and 138 ookinetes per µl per oocyst formed in feedings with purified ookinete cultures, representing a 3-fold reduction in infectivity. Likewise, the infection prevalence obtained here with purified ookinetes was, on average, 10% higher in comparison to the cited study, while the infection prevalence obtained with non-purified ookinetes was approximately the same.These results are also comparable to those obtained by Sinden R.E., et al., (2007) with non-purified ookinetes.