organism: Plasmodium berghei
attributes.tag: source_name
attributes.value: ANKA
attributes.tag: Stage
attributes.value: Hepatic merozoite
attributes.tag: chip antibody
attributes.value: none
attributes.tag: strain
attributes.value: ANKA
attributes.tag: isolation
attributes.value: HepG2 liver cells were cultured according to standard practices in complete DMEM medium (Dulbecco's Modified Eagle Medium supplemented with 10% (v/v) fetal bovine serum, 1X glutaMAX, 100 U/ml penicillin, and 100 µg/ml streptomycin) at 37°C and 5% CO2. The day prior to infection, HepG2 cells were seeded at 75,000 cells per well in a volume of 500 µl of complete DMEM in a 24-well plate. The day of infection, salivary glands containing P. berghei sporozoites were dissected from live mosquitos (obtained from the University of Georgia insectary, GA, USA) and ground with a small pellet pestle to release sporozoites. Released sporozoites were filtered through a 40 µm filter and counted using a hemocytometer. Sporozoites were diluted to 40,000 sporozoites per 300 µl in infection medium (complete DMEM supplemented with 100 U/ml penicillin, 50 µg/ml streptomycin, and 150 µlg/ml neomycin and 50 µg/ml kanamycin, and 7.5 µg/ml of the antifungal amphotericin B. Culture medium was removed from the wells containing the adherent HepG2 cells and 300 µl of the sporozoite dilution was added to each well. The plate was centrifuged at 2,000 × g for 5 min. at RT. Two hours post infection, all medium was removed and replaced with 1 ml infection medium. At 48 hpi the medium replaced once more with 1 ml fresh infection medium. Infected cells were cultured under normal conditions until merosomes were harvested 68 – 75.5 hpi. To harvest merosomes, the culture medium was collected in a 15-ml tube. Next, the empty wells were rinsed with 500 µl of infection medium and this medium was added to the original collection. Merosomes were pelleted by centrifugation at 4,000 × g for 5 min. at RT. Supernatant was removed until roughly 100 µl remained. The merosome pellet was resuspended in the remaining supernatant and transferred to a 1.5-ml tube. PBS was added to the merosomes to a final volume of 1 ml. Merozoites were released from merosomes by incubation with 1% saponin in milliQ water for 2 min. at RT. Merozoites were pelleted by centrifugation at 4,000 × g for 4 min. at RT. Supernatant was removed until roughly 100 µl remained, and merozoites were resuspended in the remaining medium. PBS was added to the resuspended merozoites to a final volume of 1 ml.
attributes.tag: geo_loc_name
attributes.value: missing
attributes.tag: collection_date
attributes.value: missing