eoe sub articles

EoE Sub Articles

1. In Vitro Purification of Sporozoites from C. parvum Oocysts
<ol>
	<li>Prepare Cryptosporidium oocysts for infecting cells (Figure 1A). 
	NOTE: Cryptosporidium oocysts were purchased from a commercial source (see the Table of Materials). These oocysts are produced in calves and are stored in phosphate-buffered saline (PBS) with antibiotics. They can be stored for about 3 months at 4 &#176;C and should ...

in vitro sporozoite isolation from cryptosporidium parvum oocysts

Source: Dutta, D. et al., <a href="https://app.jove.com/v/59610">Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection.</a> J. Vis. Exp. (2019)
In this video, we demonstrate an in vitro method to purify sporozoites from thick-walled oocysts of the pathogenic parasite Cryptosporidium parvum. The isolated sporozoites can be used for infection studies.

<img alt="Figure 1" src="/files/ftp_upload/21719/21719_Figure1.jpg" /> 
Figure 1:&#160;Preparation and purification of&#160;Cryptosporidium&#160;oocysts and sporozoites. (A) Schematic representation of the method used for oocyst and sporozoite preparation for infection. (B) Image showing&#160;in vitro&#160;excystation of oocysts. Filtration of unexcysted oocysts and shells gives a purified solution of sporozoites. Scale bar = 10 &#181;m.

In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts

1. In Vitro Purification of Sporozoites from C. parvum Oocysts

	Prepare Cryptosporidium oocysts for infecting cells (Figure 1A).
	NOTE: Cryptosporidium ...

To isolate sporozoites from oocysts of Cryptosporidium parvum in vitro, take bleach-treated oocyst suspension.
Add excystation media containing sodium taurocholate and trypsin.
Sodium taurocholate weakens the glycocalyx layer and oocyst walls, while trypsin degrades oocyst wall proteins, resulting in oocyst excystation and sporozoite release.
Microscopically assess the excystation efficiency.
Centrifuge to pellet the sporozoites, intact oocysts, and oocyst shells.
Resuspend the pellet and pass it through a filtering assembly placed on ice to keep the sporozoites dormant.
Larger oocyst shells and unruptured oocysts are retained in the membrane filter, while small-sized sporozoites pass through the filter via gravity and are collected in the filtrate.
Centrifuge the filtrate.
Resuspend the sporozoite pellet in media containing a dye and reducing buffer constituents.
The dye aids in visualizing the sporozoite injection procedure, while the reducing buffer creates a gastrointestinal tract-like environment, enhancing sporozoite viability and infectivity.
The prepared sporozoites are ready for injection procedures.

In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts

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