35 mm coverslip dishes

HFF cells were grown to confluency in 35 mm coverslip dishes (MatTek, Ashland MA, USA). RH-GFP T. gondii [29 (link)] were harvested by filtering freshly lysed parasites. Parasites were diluted 4-fold in D10+ media. After 30 min of invasion at 37 °C, the cell monolayer was washed with PBS to remove extracellular parasites. D10+ media alone, D10+ with DMSO, or drug in DMSO was added to the dishes. Actively growing parasites in fibroblasts were imaged every 12 h over a 48-hour period using a Zeiss Axiovert 200 M and an Axiovision camera to collect 10 random fields of view for each condition. Parasite replication states were manually quantified and tallied to enumerate singlets, doublets, quadruplets, and larger rosettes. Data are displayed as the fraction of total vacuoles/field that contain singlet, doublet, quadruplet, or larger (8+) numbers at the 36-hour time point.

RBL-2H3 mast cells were maintained in monolayer culture through weekly passage as described previously (Gosse et al., 2005 (link)). For stimulation, cells were sensitized with 1 μg/ml anti-DNP IgE (Gosse et al., 2005 (link)) for 2-24 h. For transfection, cells were sparsely plated (1-3×10⁵/ml) in six-well plates for fluorimetry experiments, or on #1.5 coverslips or in 35 mm cover slip dishes (MatTek Corporation, Ashland, USA) for confocal imaging. After overnight culture, cells were transfected using 2.5 μg DNA and 10 μl FuGENE HD (Promega) in 1 ml OptiMEM per well for 3-4 h in the presence of 1 ng/ml phorbol 12,13-dibutyrate to enhance DNA uptake for RBL cells (Gosse et al., 2005 (link)). Samples were then washed into full media and cultured for 16-24 h to allow for protein expression.
Jurkat T cells were maintained in suspension culture in RPMI medium with 10% fetal bovine serum and 1 μg/ml gentamicin. These cells were transfected using TransIT/Jurkat from Mirus Corporation for 16-24 h per manufacturer's recommendations.
Cells were then washed in buffered salt solution (BSS: 135 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5.6 mM glucose, 20 mM HEPES, pH 7.4) with 1 mg/ml bovine serum albumin for experiments with short chain ceramides.