Fatty acid uptake assay

The uptake of fatty acid into PHT cells was measured using the fluorescent long chain fatty acid analog C₁-BODIPY_500/510-C₁₂ (Molecular Probes). BODIPY uptake medium consisted of 80 µM fatty acid-free BSA and 2.5 nM BODIPY in Hanks balanced salt solution. PHT cells were cultured in six-well plates for 24 hours under standard conditions, and then an additional 24 hours in either standard or hypoxic atmospheres. The cells were then incubated 1 hour at 37°C in serum-free DMEM, rinsed, and 800 µL of transport medium was added to each well and incubated at RT for 3 minutes. The uptake was stopped by the addition of 3.5 mL/well ice-cold stop solution [0.1% fatty acid-free BSA, 500 µM phloretin (Sigma) in PBS]. The cells were rinsed, lysed, and fluorescence measured on a Packard FluoroCount Microplate Fluorometer and expressed as relative fluorescence units. Nonspecific association of labeled fatty acid with trophoblasts was determined using duplicate cultures assayed in the presence of 500 µM phloretin, which blocks receptor-mediated fatty acid transport and was included in the preincubation, prewash, and fatty acid transport buffers in parallel cultures. Relative fluorescence was normalized to nuclei that were stained for 3 minutes with 0.1 mg/mL DAPI in PBS and quantified as noted earlier.