1 mM EDTA, and 55Fe radioactivity determined in the upper and lower chamber buffers and the cell layer. ROS measurement To determine if compound affected cellular production of ROS, 5 × 105 K562 cells were washed, treated for 30 min with compound in Hepes-NaCl buffer, and intracellular levels of ROS detected with CM-H2DCFDA by flow cytometry as described . ROS levels are presented as mean fluorescence PF-3084014 intensity in the appropriate gated areas. K562 cells exposed to 10 μM H2O2 were used as positive control for ROS generation. Cell proliferation and colony formation assays To assess cell proliferation PC-3 cells were seeded into 96-well plates at 1 × 104/well for 24 hr to allow
for cell attachment. Cells were treated with 0.1% DMSO, 10 μM ferric ammonium citrate, 10 μM LS081, or the combination of 10 μM Fe + 10 μM LS081 in RPMI1640-10% FCS for 24-72 hr with the treatment media being find more replenished every 24 hr. Cell proliferation was accessed 24, 48, or 72 hr after treatment. In separate experiments, PC-3 or 267B1 cells were plated in 96-well plates at 1 × 104/well in RPMI1640 containing 10% FCS overnight before 24 hr treatment with 0.1% DMSO, 2 μM ferric ammonium citrate, 3 or 10 μM LS081 ± Fe in serum-free-RPMI1640, with an additional 24 hr incubation in RPMI-1640-10%
find protocol FCS without LS081. Cell proliferation was assayed with CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega) kit on a Synergy 2 Spectrophotometric Analyzer (BioTek Inc., Winooski, Vermont) with wavelength of 490 nM and the results standardized to the percentage of inhibition induced by DMSO alone. Cell viability was assessed by Trypan blue exclusion. Colony formation was assayed in PC-3 cells by plating 500 cells/well in 6-well plates in 10% FCS-RPMI1640 for 48 hr, followed by incubation with 0.1% DMSO, 10 μM ferric ammonium citrate, 3 or 10 μM LS081 ± ferric ammonium citrate for an additional 48 hours, after which the media was replaced with 10% FCS-RPMI1640. The cells were cultured for an additional 10-14 days and then stained with Crystal violet
before colonies consisting Buspirone HCl of more than 50 cells were enumerated. Results A cell based fluorescence assay to screen small molecules that increase iron transport into cells We utilized an intracellular calcein fluorescence screening method modified from Brown et al.  to screen a library consisting of ~11000 small molecules for their ability to increase or decrease iron uptake into cells. As noted in the Method, compounds which enhanced the calcein fluorescence quenching induced by iron were considered to be iron facilitators while those that decreased fluorescence quenching were considered inhibitors of iron uptake. In the initial screening of the compounds obtained from ChemDiv thirty compounds exhibited negative values for Δ Fn, i.e.