TITLE: Ookinete purification by centrifugation
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 first exploits the ookinete's biological property of laking a Cl</span><span style = "vertical-align:super;">-</span><span>/HCO</span><span style = "vertical-align:sub;vertical-align:sub;">3</span><span style = "vertical-align:sub;vertical-align:sub;">-</span><span> channel in the membrane. On the contrary, the red blood cells (RBC) do have this channel. Because of this, RBC can be selectively lysed using an isotonic solution of NH</span><span style = "vertical-align:sub;">4</span><span>Cl. In solution NH</span><span style = "vertical-align:sub;">4</span><span>Cl dissociates into NH</span><span style = "vertical-align:super;vertical-align:sub;">4</span><span style = "vertical-align:super;vertical-align:sub;">+</span><span> and Cl</span><span style = "vertical-align:sub;">-</span><span>, the NH</span><span style = "vertical-align:sub;vertical-align:sub;">4</span><span style = "vertical-align:sub;vertical-align:sub;">+</span><span> then transforms into NH</span><span style = "vertical-align:super;">3</span><span>, which is permeable to the plasma membrane in contrast to the NH</span><span style = "vertical-align:super;vertical-align:sub;">4</span><span style = "vertical-align:super;vertical-align:sub;">+</span><span>. Once inside the cell, NH</span><span style = "vertical-align:super;">3</span><span> establishes an equilibrium with NH</span><span style = "vertical-align:super;vertical-align:sub;">4</span><span style = "vertical-align:super;vertical-align:sub;">+</span><span> resulting in a net influx of Cl</span><span style = "vertical-align:sub;">-</span><span> through the aforementioned ion channel, increasing the NH</span><span style = "vertical-align:super;">4</span><span>Cl concentration in the RBC considerably and, therefore, of water, causing an osmotic shock and RBC burst. Since HCO</span><span style = "vertical-align:super;vertical-align:sub;">3</span><span style = "vertical-align:super;vertical-align:sub;">-</span><span> is in equilibrium with CO</span><span style = "vertical-align:super;">2</span><span>, RBC lysis must be performed during shaking to increase the concentration of CO</span><span style = "vertical-align:sub;">2</span><span> in the solution (Chernyshev, et al., 2007).</span></div><div class = "text-block">Next a series of differential centrifugations are performed exploiting the sedimentation rate of the ookinetes, which depends on the size, shape, and density of the ookinetes as well as on the viscosity of the medium. The last step involves isopycnic centrifugation in a density cushion taking advantage of the buoyant density of the ookinetes.</div><div class = "text-block">This method has been modified from Rodríguez M.C., et al., 2002 and Carter V., et al., 2003.</div></div>

[STEPS]
?. Take the ookinete culture form the incubator and mix well to re-suspend the sedimented cells.Transfer to a 50 ml conical-bottom tube and
Centrifuge: 1500 1324, 3 min, 20 10
?. Remove supernatant and add NH4Cl at a ratio of 1:40 in relation to the volume of the pellet.Transfer the solution into a 500 ml Erlenmeyer flask.
First, add 1 or 2 ml of NH4Cl and pipette up and down vigorously to disaggregate the pellet. Then add the rest of the solution (about 40 ml in total).
20 33
?. Transfer the solution into a 50 ml conical-bottom tube and  .
Centrifuge: 725 1324, 3 min, 20 10
?. Remove the supernatant and add  of PBS.Pipette vigorously to disaggregate the pellet.Then add  of PBS.
2 mL
40 mL
Centrifuge: 500 1324, 3 min, 20 10
Ookinetes are very sticky and in the absence of a substrate tend to form clumps that greatly reduce the outcome of the purification.It is important to pipette thoroughly and to add only one ookinete culture per centrifuge tube.
?. Repeat step 4 but with the following centrifuge conditions:  .
Centrifuge: 270 1324, 5 min, 20 10
?. Remove supernatant, add  of PBS, and pipette vigorously.
2 mL
?. Prepare  of Nycodenz at 17% with iohexol buffer in a 15 ml conical-bottom tube and slowly add the  of ookinetes-PBS on the iohexol cushion.Avoid mixing the two solutions.
5 mL
2 mL
Centrifuge: 895 1324, 15 min, 20 10
?. Remove the brownish interphase between the PBS and the iohexol that contains the purified ookinetes.Transfer the interphase to a 15 ml conical-bottom and add  of PBS.
10 mL
?. to remove the Nycodenz. Repeat two times.
Centrifuge: 270 1324, 5 min, 20 10