Quantitative Tracking of Cryptosporidium Infection in Cell Culture With Cfse

Abstract

Immunofluorescence-based assays have been developed to detect and quantitate Cryptosporidium parvum infection in cell culture. Here, we describe a method that tracks and quantifies the early phase of attachment and invasion of C. parvum sporozoites using a fluorescent dye. Newly excysted sporozoites were labeled with the amine-reactive fluorescein probe carboxyfluorescein diacetate succinimidyl esters (CFSE) using an optimized protocol. The initial invasion of cells by labeled parasites was detected with fluorescent or confocal microscopy. The infection of cells was quantified by flow cytometry. Comparative analysis of infection of cells with CFSE-labeled and unlabeled sporozoites showed that the infectivity of C. parvum was not affected by CFSE labeling. Quantitative analysis showed that C. parvum Iowa and MD isolates were considerably more invasive than Cryptosporidium hominis isolate TU502. Unlike immunofluorescent assays, CFSE labeling permitted the tracking of the initial invasion of C. parvum. Such an assay may be useful for studying the dynamics of host cell-parasite interaction and possibly for drug screening.

Fluorescent Eimeria bovis sporozoites and meront stages in vitro: a helpful tool to study parasite–host cell interactions

“…Corresponding results were reported with CFSE-stained Cryptosporidium parvum and Cryptosporidium hominis sporozoites (Feng et al 2006). Also in accordance with our results, the authors demonstrated that CFSE-stained C. parvum sporozoites discharge fluorescent material during gliding motility.…”

“…It has not been observed when Cryptosporidium spp. sporozoites were stained (Feng et al 2006). The dye binds to aminomoieties (McCabe et al 2002) and therefore should predominantly react with basic proteins, particularly to those with many moieties, e. g. histones, hyaluronates, glycosaminglycans or amino sugars (Suniara et al 2000;Spadaro et al 2007).…”

“…This staining was recently shown to be useful for tracing Cryptosporidium spp. sporozoites in vitro (Feng et al 2006). …”

The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans

“…For attachment assays, hypochlorite-treated oocysts were incubated in 0.75% sodium taurocholate in PBS for 30 min at 37°C. Excysted sporozoites were labeled with 10 M Vybrant carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) (28). Sporozoites were purified by filtration through a 3-m filter (Millipore, Billerica, MA).…”

Human CD8+ T Cells Clear Cryptosporidium parvum from Infected Intestinal Epithelial Cells

“…Parasites were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) according to a previously published protocol with modifications. 17 After 15 min of incubation in PBS/0.8% Na-taurocholate, 6 μL of CFSE solution (Invitrogen, Carlsbad, CA) were added, and parasites were incubated for another 30 min. Parasites were washed and added to 0.…”