Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds.

Experimental compounds and reagents

As proof of principle, eighty experimental chemical compounds were used in the present study which were included in a single plate (Plate 60216) that was randomly chosen from the DIVERSet small molecule library (Code NT797; ChemBridge Corporation, San Diego, CA). The ChemBridge ID number, chemical structure, molecular weight and molecular name of the experimental chemical compounds are available upon request. Compound stock solutions were received already dissolved in Dimethyl sulfoxide (DMSO) and dilutions of each of them were added directly to individual wells containing target cells in complete culture media. Hoechst 33342 (Hoechst; Invitrogen, Eugene, OR); and Propidium iodide (PI; MP Biomedicals, Solon, OH) stock solutions were mixed, diluted in cell culture media and added to each experimental well at a final concentration of 1 μg/ml for each dye.

Cell lines and culture conditions

Two cell lines were utilized in the implementation of this DNS assay, the adherent NIH 3T3 murine fibroblast cells (American Type Culture Collection, Manassas, VA; CRL-1658), and the non-adherent YT human lymphoma cells ( Yodoi et al. 1985 ). The culture media for adherent cells was DMEM (HyClone, Logan, UT) plus 10% heat-inactivated newborn calf serum (HyClone), while that for non-adherent cells was RPMI (HyClone) media containing 10% heat inactivated fetal bovine serum (HyClone). Both culture media were supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Lonza, Walkersville, MD). The incubation conditions of the cells were at 37°C in humidified 5% carbon dioxide (CO 2 ) atmosphere, in a regular water jacketed incubator or the environmentally controlled chamber of the bioimaging system. To prepare the experimental multiwell plates and to guarantee high viability, adherent cells were washed with fresh media to eliminate cell debris and floating cells consisting mainly of dead cells. When necessary, the viability of non-adherent cells was increased by using Ficoll-Paque ™ PLUS density gradient centrifugation ( Boyum 1968 ). Briefly, after centrifugation at 400xg for 30 min at room temperature, live cells at the interface were collected and washed with fresh culture media. Subsequently, these non-adherent cells were expanded by starting a new culture. Only cultures containing cell viabilities of 95% or higher were used for the cytotoxicity studies. NIH 3T3 were harvested from culture flasks by adding 2 ml of 0.25% trypsin solution (Invitrogen, Carlsbad, CA), diluted in serum free DMEM, and incubated for approximately 5 min at 37°C. Trypsin was neutralized by addition of culture media with 10% serum. NIH 3T3 and YT cells were seeded at a cell density of 15,000 and 10,000 cells/well, respectively, in glass-bottomed 96-well plates (BD Biosciences, Rockville, MD) using 200 μl of culture media/well. When the 384-well plate format (BD Biosciences) was utilized, NIH 3T3 and YT cells were seeded at a density of 3,750 and 2,500 cells per well in a volume of 50 μl culture media, respectively. For the MTS assay, NIH 3T3 cells were seeded at 15,000 cells in 100 μl of media culture per well in a flat-bottomed plastic 96-well plate (Falcon, Franklin Lakes, NJ). Cell seeding was achieved with a robotic pipette system (epMotion 5070, Eppendorf, New York, NY). Multiwell plates containing cells were incubated overnight in culture media prior to addition of the chemical compounds. Image acquisition and analysis The BD Pathway 855 bioimager system and its associated AttoVision v1.6.2 software (BD Biosciences, Rockville, MD) were utilized for image collection and analysis. After dye addition and incubation, images were captured with two software predetermined excitation/emission filter sets; 380/535 nm for Hoechst and 555/645 nm for PI. Images from each well were acquired using a 20x/NA 0.75 dry objective. In order to obtain sufficient cell numbers (ROIs) for statistically robust data, images from four contiguous fields (2x2 montage) were obtained per well. Under these settings and by plating NIH 3T3 and YT cells at the cell densities indicated above, the system was able to capture from ~100 to 400 cells per well and the images were subsequently analyzed using the AttoVision software. To define nuclei as individual units or regions of interest (ROIs), pre-processing filters and intensity thresholds were applied for image segmentation. Segmented images were subjected to data classification by the use of the AttoVision software. The percentage of dead cells was calculated from the total number of ROIs per well. Cell nuclei emitting fluorescence signal from both Hoechst and PI (fluorescence co-localization) were considered as dead cells, while cells emitting only Hoechst signal were counted as live cells. Data was classified by the use of the AttoVision software to identify compounds with cytotoxic activity and hits were identified by applying the 2xSD principle (see below). Validation of the DNS assay with known toxic and non-toxic compounds Validation of the DNS assay was performed utilizing NIH 3T3 cells, 96-well plates, and a panel of 10 compounds with known toxic or non-toxic activity. Three antibiotics: ampicillin (Fisher Scientific, Pittsburgh, PA), kanamycin (Fisher Scientific) and streptomycin (Fisher Scientific) were included as non-toxic compounds at a final concentration of 100 μg/ml. Also, seven compounds that cause toxic effects on mammalian cells were utilized at final concentrations indicated as follows: 100 μM Cisplatin (Strem Chemicals, Newburyport, MA), 1444 μM Geneticin (G418; Sigma-Aldrich, Inc., St Louis, MO), 379 μM hygromycin B (EMD Chemicals, Gibbstown, NJ), 10 μM iodoacetamide (Sigma), 20 μM plumbagin (Sigma), 500 μM titanocene (Sigma), and 0.1% v/v Tween 20 (Acros Organics, New Jersey). An initial experiment included all ten compounds incubated for 20h, to detect those with fast activity. A second experiment extended the incubation time for 72h and 120h in order to detect the cytotoxic activity of Cisplatin, Geneticin, hydromycin B and iodoacetamide, which display a slow cytotoxic effect. Cell culture conditions, cell density, compound concentration, image acquisition and analysis were the same as described above. Evaluation of the DNS assay robustness The Z′ factor value for the DNS assay was calculated over 48 data points for 96-well plates using NIH 3T3 and YT cells prepared as described above. Z′ factor assays were carried out utilizing Hydrogen peroxide (H 2 O 2 ; Acros Organics, NJ) as a positive control for cytotoxicity at a final concentration of 300 μM ( Miyoshi et al. 2006 ; Zierdt 1986 ) for 20h. Ninety-six data points for H 2 O 2 treated as well as for untreated cells were used to calculate the Z′ factor values in the 384-well plate format. Dye addition and image acquisition were perform as described above. The following equations were used to calculate the Z′ factor value, S/N and S/B ratio values: Z′ factor, Z′=1−(3σ T +3σ U )/(μ T −μ U ); signal-to-noise, S/N= (μ T −μ U )/σ U ; signal-to-background, S/B= μ T/ μ U ( Zhang et al 1999 ); and the coefficient of variation percent value (%CV) for treated cells; %CV= (σ T /μ T ) 100 ( Inglese et al. 2007 ). Where σ T is the standard deviation of the wells corresponding to the treated cells, σ U is the standard deviation of the untreated controls, μ T is the mean value of the wells corresponding to the treated cells, and μ U is the mean value of the untreated controls.

Primary screening using the DNS assay

As a primary screen, experimental compounds were tested for their cytotoxicity on the NIH 3T3 cell line at a final concentration of 100 μM in the presence of 1% v/v DMSO per well and dispensed into the wells via a robotic pipetting system (epMotion 5070). As positive controls for cytotoxicity, cells treated with 300 μM H 2 O 2 were included in each experimental plate. DMSO (Sigma), the solvent used for all chemical compounds, was added at a final concentration of 1% v/v and used for normalization purposes to account for any DMSO-induced cytotoxicity (which was negligible). Untreated cells were included in all the experiments to establish the background of dead cells provoked by cell manipulation and factors inherent to the culture system. Cells exposed to the experimental chemical compounds and the controls were incubated for a total of 20h under the conditions described above. One hour prior to image capture, the mixture of the two fluorescent dyes was dispensed into the experimental wells by the use of the onboard pipetting system of the bioimager. To discern between cytotoxic and non-cytotoxic compounds, a threshold of 2 times standard deviation (2xSD) from the mean percentage of dead cells from all the samples was applied ( Malo et al. 2006 ) as described below.

Primary screening using the MTS assay

To establish a comparison between the DNS and MTS assays, NIH 3T3 cells were utilized at the same density as indicated above. The same 80 chemical compounds were tested at the same final concentration (100 μM) utilized with the DNS assay. After 18h of incubation, 20 μl of the MTS reagent (CellTiter 96 AQueousOne Solution Cell Proliferation Assay; Promega, Madison, WI) were added to each well and subsequently incubated for an additional 2h, completing 20h of incubation. The colored formazan product was measured by absorbance at 490 nm with a reference wavelength of 650 nm using a microplate reader (VERSA max microplate reader, MDS, Inc., Toronto, Canada). Control wells without cells and containing the same volumes of culture medium and MTS reagent were used as blank to subtract background absorbance. Data were expressed as percentage of cell viability relative to DMSO (solvent control) treated cells. However, in order to make the data comparable to the DNS results, the percentages of dead cells were obtained by subtracting the percentage of cell viability from 100. In this assay the same 2xSD rule (see following section) was applied to determine whether a compound was cytotoxic or not.

Cut-off value for cytotoxicity in primary screening assays

The cut-off values for the DNS and MTS primary screening assays were determined by the 2xSD rule ( Malo et al 2006 ), where the hit threshold is defined as 2xSD beyond the mean of the screened samples. Therefore, compounds whose measured cytotoxicity were equal or exceeded the hit threshold were considered as hits. In order to determine comparable primary screening cut-off values, the 2xSD value was independently calculated for both DNS and MTS assays. Since the DNS assay was run three independent times to determine its reproducibility, the cut-off value was calculated as the mean of the individual 2xSD values derived from each experiment.

Secondary screening for hit validation

To further validate the cytotoxicity of the identified hits from the primary screening on NIH 3T3 cells, cell death was analyzed by the DNS and a flow cytometry assay. NIH 3T3 and YT cell lines were treated with a single concentration (100 μM) of chemical compound in triplicate. Cell density, culture conditions and image acquisition were performed as described above. Cell death was evaluated via flow cytometry using the PI staining method as previously described ( Varela-Ramirez et al. 2011 ). YT and NIH 3T3 cells were seeded in a 96-well plate as described above and treated with test compounds for 20h. After incubation, floating NIH 3T3 cells were transferred to an ice-cold tube, while adhered cells were treated for 5 min with 0.25% of trypsin solution. Cells from each individual well, including those floating and those harvested by trypsinization, were washed with cold complete media and cold PBS, pelleted by centrifugation (1,400 rpm for 5 min at 4°C) and resuspended in 300 μl of PBS containing 10 μg/ml of PI ( Shaik et al. 2009 ). The same protocol omitting the trypsinization step was applied for the YT cells. Cells were gently vortexed and immediately analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter, Miami, FL). For each sample, a maximum of 3,000 individual events were collected and analyzed using CXP software (Beckman Coulter, Miami, FL). All compounds were analyzed in triplicate and cytotoxicity was determined as the percentage of PI stained cells. Generation of dose-response curves and CC 50 values Glass-bottomed 96-well plates were prepared and processed as described above using NIH 3T3 cells. Chemical compounds identified as hits were tested at several concentrations to create dose-response curves and determine the 50% cytotoxic concentration (CC 50 ). CC 50 was defined as the compound concentration causing loss of membrane integrity of 50% of the cell population as compared to solvent treated cells after 20h of incubation. The CC 50 values were obtained from linear portions of the dose-response curves as previously described ( Varela-Ramirez et al 2011 ). Briefly, data was normalized by subtracting from each experimental value the average percentage of dead cells from six wells treated with 1% v/v DMSO (solvent control). The two compound concentrations closest to the 50% cytotoxicity value were plotted using linear regression and the CC 50 values were extrapolated from the line equation.

Experimental compounds and reagents

As proof of principle, eighty experimental chemical compounds were used in the present study which were included in a single plate (Plate 60216) that was randomly chosen from the DIVERSet small molecule library (Code NT797; ChemBridge Corporation, San Diego, CA). The ChemBridge ID number, chemical structure, molecular weight and molecular name of the experimental chemical compounds are available upon request. Compound stock solutions were received already dissolved in Dimethyl sulfoxide (DMSO) and dilutions of each of them were added directly to individual wells containing target cells in complete culture media. Hoechst 33342 (Hoechst; Invitrogen, Eugene, OR); and Propidium iodide (PI; MP Biomedicals, Solon, OH) stock solutions were mixed, diluted in cell culture media and added to each experimental well at a final concentration of 1 μg/ml for each dye.