Glass bottom dark 24 well plates

HFFs were cultured on glass-bottom dark 24-well plates (Greiner Bio-One) and subsequently infected with tachyzoites for a 24-h period in regular growth media. Afterward, the cells were rinsed with PBS, and the growth medium minus phenol red (GMPR) supplemented with 10-µM 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDCFDA) was added to the cells for a 10-min incubation at 37°C. CDCFDA was prepared by diluting it sequentially into GMPR from a 10-mM imethyl sulfoxide (DMSO) solution. The media containing the dye was then removed, and the cells were washed three times with PBS before being replenished with GMPR. Immediately after, the cells were subjected to imaging. At least 50 vacuoles per well were quantified and classified as either CDCFDA positive or CDCFDA negative.

HFFs were grown on glass-bottom dark 24-well plates (Greiner Bio-One) and infected with tachyzoites for 24 h in regular media. The cells were washed with PBS and medium was replaced with DMEM plus 10% FBS minus phenol red (GMPR) supplemented with 10 μM 5(6)-Carboxy-2′,7′-dichlorofluorescein diacetate (CDCFDA) for 10 min at 37°C. CDCFDA was sequentially diluted into GMPR from a 10 mM DMSO solution. The dye-containing media was removed, and the cells were washed three times with PBS, replaced with GMPR, and were immediately imaged.

HFFs were grown on glass-bottom dark 24-well plates (Greiner Bio-One) and the confluent monolayers were infected with different parasite strains at an MOI of 1 in DMEM containing 1% FBS. At 24 h post-infection, the cells were washed with PBS after which Gibco DMEM/F-12 (Invitrogen) medium without phenol red and supplemented with 10 μM 5(6)-Carboxy-2’,7’-dichlorofluorescein diacetate (CDCFDA) was added to the cells. After 10 minutes of incubation at 37°C, the medium was removed and the cells were washed three times with PBS. Gibco DMEM/F-12 growth medium was added to the cells and the cells were imaged immediately.