TITLE: Affinity purification of ookinetes in coverslips 
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 coverslip allows the obtention of highly pure ookinete cultures that are ready for oocyst culture or further experimentation while still attached to the ECM gel coating.</div></div>

[STEPS]
?. [Purification from cardiac puncture-derived ookinete culture.]
Thaw an aliquot of 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
?. [Purification from cardiac puncture-derived ookinete culture.]
Place the desired number of clean and sterile coverslips in the wells of a 24-well culture plate.Coat the coverslips with   of ECM gel and allow gel formation at   for  .
12 µl
37 °C
Avoid spilling the ECM gel outside of the coverslip.Optionally, the coated coverslips 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 500 µl of ookinete medium for 30 min at 37°C. Remove the medium before adding the ookinete culture.
?. [Purification from cardiac puncture-derived ookinete culture.]
Add between    and   of the ookinete culture per coverslip and incubate at   for  .
0.5 mL
3 mL
20 °C
?. [Purification from cardiac puncture-derived ookinete culture.]
15 min before the incubation ends, tilt the culture plate at a 10-15° angle. This will cause the sedimented cells to slide to one side of the container leaving most of the coverslip already free of contaminant cells.
?. [Purification from cardiac puncture-derived ookinete culture.]
Prepare another 24-well culture plate with   of ookinete or oocyst medium.
1 mL
?. [Purification from cardiac puncture-derived ookinete culture.]
Lift a coverslip with the needle hook and grab it with the tweezers.While holding the coverslip in a vertical position, wash it with   of PBS.One or two washes is enough to obtain ookinete purities over 85%.
1 mL
Once the coverslips are removed from the wells and PBS is added, the ookinete culture will start to agglutinate. It is best to dip the coverslip about 1/3 or 1/4 of its diameter into the ookinete culture a couple of times to lose the majority of the contaminant cells before washing with PBS.
?. [Purification from cardiac puncture-derived ookinete culture.]
Transfer the coverslip to the new 24-well culture plate previously filled with medium.
?. [Purification from cardiac puncture-derived ookinete culture.]
Repeat steps 6 and 7 for all the coverslips.
?. [Purification from cardiac puncture-derived ookinete culture.]
The leftover ookinete culture can be used to perform another purification by placing it on new coated coverslips.
?. [Purification from microcultures.]
This protocol can also be used to obtain purified ookinetes derived from microcultures using only 1 µl of infected blood.Obtain 1 µl of tail blood of an infected mouse as explained in the Plasmodium berghei Ookinete Culture protocol and place in 0.6 Eppendorf tubes with 7 µl of ookinete culture and 1 µl of heparin.Incubate for 18 h at 20°C.
?. [Purification from microcultures.]
Prepare the coated coverslips as explained in steps 1 and 2.
?. [Purification from microcultures.]
Bring the microculture to 2 ml with ookinete medium and add it to a coated coverslip.
?. [Purification from microcultures.]
Repeat steps 4 to 8 to obtain the purified ookinetes.
Starting from microcultures, approximately 297 ookinetes can be obtained per coverslip.