multiple survival strategies that are usually the hallmark of tumor development and progression. Therefore, development of potent and selective chemical probes that can modulate ABCB6 transporter function may have oncologic as well as pharmacologic applications. Development of such modulators would also improve our understanding of ABCB6 substrate specificity and ABCB6 transporter function with regards to heme biosynthesis, mitochon- drial function, and cellular adaptation to metabolic demand and stress. In this report, we describe the development, optimization, and validation of a novel robust high-throughput fluorescence based flow cytometry assay designed to interrogate modulators of ABCB6 activity. We have previously demonstrated that ABCB6 regulates the synthesis and accumulation of the fluorescent compound protoporphyrin (PPIX) [14,17]. Increased ABCB6 expression in cells selected for over-expression of wild-type ABCB6 results in increased cell-associated PPIX fluorescence intensity while having no effect on the fluorescence properties of the molecule [14,17]. This has formed the basis of a flow cytometry assay to develop modulators of ABCB6 activity. Using this assay, we have identified and characterized modulators of ABCB6 activity from the Prestwick Chemical Library (PCL). The identified chemotypes may represent leads for the development of novel chemical probes for ABCB6. Materials and Methods Cells Lines and Culture Conditions Human Erythroleukemia (K562) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). K562 cells were engineered to constitutively express either the wild-type ABCB6 or the mutant ABCB6 (under the control of a CMV promoter) as described [14,17]. These cells exhibit stable expression of ABCB6 for 60 passages (Fig. S1). Cells were cultured as previously described [14] using modified eagle’s medium (CellGro, VA) supplemented with 10% fetal bovine serum (Hyclone, Logan UT) and 100 units/mL penicillin/streptomycin. Chemicals The Prestwick Chemical Library (http://www.prestwickchemical.fr/) contains 1120 small molecules, 90% marketed drugs and 10% bioactive alkaloids or related substances with a high degree of drug- likeliness. The active compounds were selected for chemical and pharmacological diversity and their potential for clinical trial drug repurposing since their bioavailability and safety is well document- ed. The PCL 2000 H was designed to reduce the risk of ‘‘low quality’’ hits, reduce the cost of the initial screening, and accelerate lead discovery. The 1120 molecule collection of the library supersedes a previous 880 small molecule collection. Tomatine, benzethonium chloride (BEC), verteporfin, piperlongumine, and succinylacetone were subsequently purchased from Sigma Chemical Co (St. Louis Mo). HTS Flow Cytometry and Data Analysis The HyperCytH flow cytometry platform [18] interfaces a flow cytometer and an autosampler that sequentially aspirates particle suspensions from individual wells or a microtiter assay plate. Between wells, the pump draws a bubble of air into the sample line generating a tandem series of bubble-separated samples for delivery to the flow cytometer. Sample and bubble volumes are determined by the time the autosampler probe is in a well or above a well taking in air. Accurate measurements have been demon- strated in endpoint assays over a range of fluorescence intensities using input cell concentrations of 1–20 million/mL and source HTS Assay for Functional Modulators of ABCB6 well volumes of 5–100 mL. Immediately after data acquisition by the flow cytometer, specialized software (IDLeQuery) was used to analyze the data file. A flow chart describing the general scheme of the HTS assay is presented in Fig. S2. General Assay and HTS Conditions A 10 mL volume of ABCB6 overexpressing or empty vector K562 cells, at a density of 4–56107 cells were transferred to 384- well microplates containing 10 mL volume of the test compounds in DMSO and 10% serum PBS. Test compounds were diluted in 5% serum-PBS-DMSO to obtain a final concentration of 6.67 mM. DMSO concentration was maintained at 1%, which is well tolerated by the cells and the assay conditions (Fig. S9). Microplates were incubated overnight at 37uC. HyperCytH sampling consisted of 1–2 mL taken from a 20 mL well volume at room temperature. Data analysis was performed with Hyper- View software program to automatically detect the time-resolved data clusters (wells) and determine the mean channel fluorescence (MCF) by FL2 (PE) [19–21]. Acquired data were automatically exported to Microsoft excel for calculating the assay quality control Z’ as described [22]. The Z’ factor is a statistical parameter utilized in high throughput screening to evaluate the ability of an assay to identify a hit compound. Z’ signal to noise (S/N), and signal to background (S/B) statistics were calculated using the following equation and as described in Zhang et al 1999. Z’ = 1 - (3*STDpos + STDneg)/(ABS[AVEpos-AVneg]). In this equation STDpos is the standard deviation of the positive controls, STDneg is the standard deviation of the negative controls, AVEpos is the mean of the positive controls and AVEneg is the mean of the negative controls. The Abosrobance (ABS) represents the absolute value of the difference between the different control means. Plate Configuration and Screening 384-well plates were configured with 32 control wells and 32 buffer wells. Columns 1 and 24 contained the 1% DMSO/buffer (negative control), columns 2 and 23 with ABCB6-mutant cells in 1% DMSO/buffer. This leaves 320 wells (columns 3 - 22) to which PCL test compounds were added. Sampling was started after incubating the plates overnight at 37uC and 5% CO2. Confirmatory Dose Response Assay Test compounds (potential hit compounds) displaying activity after the PCL screening were serially 2-fold diluted in a concentration range from 48.8 nM to 100 mM. Assay conditions were identical with the primary HTS assay employing ABCB6 overexpressing and vector cells. Triplicate curves were fitted by Prism software (GraphPad Software Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose–response model with variable slope, known as the four-parameter logistic equation. Curve fit statistics were used to determine the half- maximal effective concentration (EC50 in mM) and the Hill slope of the fitted curve. Cell Viability and Cytotoxicity The luminescent Cell Viability Assay kit CellTiter-Glo from Promega (Madison, WI) was used and the assay was performed following the manufacturer’s instruction as described previously [21]. Briefly, at the time of passage, cell suspensions (99 mL, 10,000 cells/well) were added into opaque-walled 96-well plates (Corning, Corning, NY). After stabilization, test compounds were added to the wells at 1 mL per well resulting in a final concentration range from 380 nM to 100 mM. Vehicle control PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40005