Quantitative tracking of Cryptosporidium infection in cell culture with CFSE.
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.
Feng
Hanping
H
Division of Infectious Diseases, Tufts University, Cummings School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA.
Nie
Weijia
W
Bonilla
Ruben
R
Widmer
Giovanni
G
Sheoran
Abhineet
A
Tzipori
Saul
S
D000199:Actins / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D003458:Cryptosporidium / Q000302:isolation & purification* / Q000502:physiology; D016785:Cryptosporidium parvum / Q000302:isolation & purification / Q000502:physiology; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006790:Host-Parasite Interactions; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D013194:Staining and Labeling; D013388:Succinimides
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000199:Actins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides