In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts
Published: October 31, 2023
Abstract
Source: Dutta, D. et al., Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection. 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.
Protocol
1. In Vitro Purification of Sporozoites from C. parvum Oocysts 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 °C and should …
Representative Results
Figure 1: Preparation and purification of Cryptosporidium oocysts and sporozoites. (A) Schematic representation of the method used for oocyst and sporozoite preparation for infection. (B) Image showing in vitro excystation of oocysts. Filtration of unexcysted oocysts and shells gives a purified solution of sporozoites. Scale bar = 10 µm.
Disclosures
The authors have nothing to disclose.
Materials
| EMD Millipore Isopore Polycarbonate Membrane Filters- 3µm | EMD-Millipore | TSTP02500 | |
| Fast green dye | SIGMA | F7252-5G | |
| Microfuge tube 1.5ml | Eppendorf | T9661-1000EA | |
| Micro-loader tips | Eppendorf | 612-7933 | |
| Sodium hypoclorite (use 5%) | Clorox | 50371478 | |
| Super stick slides | Waterborne, Inc | S100-3 | |
| Swinnex-25 47mm Polycarbonate filter holder | EMD-Millipore | SX0002500 | |
| Taurocholic acid sodium salt hydrate | SIGMA | T4009-5G | |
| Tween-20 | Merck | 8221840500 | |
| DMEM | Invitrogen | 12634-010 | 500ml |
| Penstrep | Gibco | 15140-122 | 5ml of stock in 500ml DMEM |
| Glutamax | Gibco | 35050038 | 5ml of stock in 500ml DMEM |
| Hepes | Gibco | 15630056 | 5ml of stock in 500ml DMEM |
| Reducing buffer (for resuspension of oocysts and sporozoites for injection) | Final concentration | ||
| L-Glutathione reduced | Sigma | G4251-10MG | 0.5 μg/μl of OME/OMD/LOM |
| Betaine | Sigma | 61962 | 0.5 μg/μl of OME/OMD/LOM |
| L-Cysteine | Sigma | 168149-2.5G | 0.5 μg/μl of OME/OMD/LOM |
| Linoleic acid | Sigma | L1376-10MG | 6.8 μg/ml of OME/OMD /LOM |
| Taurine | Sigma | T0625-10MG | 0.5 μg/μl of OME/OMD/LOM |
| INTESTINAL ORGANOID MEDIA-OMD (Differentiation media) | To differentiate organoids, expanding small intestinal organoids were grown in a Wnt-rich medium for six to seven days after splitting, and then grown in a differentiation medium (withdrawal of Wnt, nicotinamide, SB202190, in a differentiation medium (withdrawal of Wnt, nicotinamide, SB202190, prostaglandin E2 from a Wnt-rich medium or OME) |
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Cite This Article
In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts. J. Vis. Exp. (Pending Publication), e21719, doi: (2023).