BACKGROUND
high-throughput screening methods for toxicology evaluation in the early phases of drug discovery are highly desired.
in the early 1980´s a novel assay for cell survival determination was reported  <cit> , which has been frequently used to study the biological activity of a variety of potential cytostatic drugs.
the assay was presented as a rapid, precise and simple method to detect living cells in mammalian cell cultures, using the tetrazolium salt mtt - <dig> -diphenyl tetrazolium bromide) and a microtiter plate reader.
the mtt is reduced in living cells, forming formazan crystals which are then dissolved in a mixture of hcl and isopropanol  <cit> , or alternatively in dimethylsulfoxide  <cit> .
the optical density of dissolved crystals is then read in each well within the first hour.
cell survival is determined after the data analysis  <cit> .
although widely used, the mtt assay has several disadvantages: in particular poor linearity with the cell number, sensitivity to environmental conditions and more importantly the dependence of cell metabolism on formazan  <cit> .
these observations have lead to a search for different alternative assays, such as the measurement of cellular protein content with the srb dye   <cit>  and a variety of other in vitro assays  <cit> .
however, the srb assay requires a time-consuming workout, such as incubation with the dye for  <dig> min and fixation.
a faster and more reliable system able to screen large number of potential compounds in a large panel of cells would therefore be considered an enormous improvement in the pharmacology research.
the cloneselect™ imager  could offer such an opportunity.
the system was designed to be capable of screening cellular confluence and growth in a range of microwell plates.
for these measurements, the first step consists on a calibration of the cell number in the microtiter plates in used, and for each cell type.
after this step, the cells will be counted based on such calibration.
cell growth is viewed and tracked in every well, using label-free white light imaging for consistent determination of cell confluence and cell number estimation.
therefore, from such measurements the confluence in each well can be determined rapidly and efficiently.
the cloneselect imager system captures images of the entire well, which provides a label-free, non-destructive and direct analysis of the cell proliferation in each well as function of time.
moreover, the imaging of a  <dig> well plate is extremely fast, with the data acquisition in less than  <dig> min.
data can be either output into a spread sheet, or displayed visually as a series of pie charts.
the present study attempts to validate the cloneselect™ imager system for use in cellular toxicity screening of platinum-based drugs.
two different drugs have been selected for this study, namely the well-known cisplatin and oxaliplatin in two different human cancer cell lines.
a comparison is made between the mtt assay results and the results obtained using the cloneselect imager system using a bland-altman plot  <cit> .
our results unequivocally show that the cloneselect™ imager system offers a superior and rapid alternative in determining the cytotoxicity of different agents on both adherent and non-adherent cell lines.
RESULTS
plates were treated with the antiproliferative agents, imaged with the cloneselect imager system  <dig> h,  <dig> h and  <dig> h after the addition of the agents.
to compare with the mtt assay, once the plate was imaged with the cloneselect imager system, the mtt assay was performed; therefore, the results from the cloneselect™ imager and the mtt assay originate from the same plate and therefore are directly comparable.
the well-known human ovarian carcinoma cisplatin-sensitive a <dig> cells and the cisplatin-resistant counterpart a2780r cells were selected as adherent cells.
the cytotoxic activity of cisplatin and oxaliplatin has been investigated in parallel cultures under the same conditions in a pair of human ovarian carcinoma cell lines .
the incubation with the platinum compounds can be followed using the cloneselect imager system, obtaining a rapid view of the antiproliferative activity of the compounds in the cells over the time .
the cell confluence  in the wells versus time of incubation  with the cisplatin in the a2780r cells is shown in figure  <dig>
the compound was added to the cells in different concentrations to achieve a dose-response curve.
the a2780r cells were still growing  <dig> h after the addition of the compounds ; therefore, the determination of the ec <dig> value was not performed in this case.
this observation is important, since the ec <dig> value after  <dig> h of incubation with cisplatin in the a <dig> cell line has been reported  <cit> .
the same plate after  <dig> h of incubation with the platinum compound shows significant changes.
the cells incubated with higher concentrations show a lack of growth, while the cells incubated with lower concentrations show a growth comparable to the non-treated cells.
the growth inhibition of the cells is even more evident in the wells with highest concentration of the platinum compound when the same plate is imaged after  <dig> h of incubation, while the growth continue in the wells with lower concentrations of the antiproliferative agent.
similar results were obtained for cisplatin incubation in the a <dig> cells , as also observed for the incubation with oxaliplatin .
cell confluence  is also displayed directly with the cloneselect imager system, as depicted in figure  <dig>  where the two first columns  of the microtiter plate were non-treated cells.
in addition, the plate was divided to incubate the a2780r cells with both cisplatin and oxaliplatin, as described in the experimental section, using ten different concentrations.
figure  <dig> clearly shows that the cloneselect imager system provides a rapid insight into the different cytotoxic activities displayed by both platinum drugs against the cells under study.
the a2780r cells were treated with the same concentrations of cisplatin and oxaliplatin, confirming that oxaliplatin is more cytotoxic than cisplatin in the a2780r cells.
similar results as for the cytotoxicity of oxaliplatin in the a2780r cells were observed in the a <dig> cells .
moreover, it is important to mention that a different sensitivity of the a2780r cells against cisplatin was observed, compared to the sensitivity of the a <dig> cells.
for instance, in figure  <dig> the non-treated a2780r cells have a 55% confluence, while a 33% of confluence is observed when the cells are incubated with  <dig>  μm of cisplatin.
in the case of a <dig> cells  the sensitivity to the drug is higher, finding a 15% of confluence after  <dig> h of incubation with  <dig>  μm cisplatin .
comparison of cytotoxicity determined by cloneselect™ imager system and the mtt assay
to compare the cytotoxicity results observed with the cloneselect™ imager system and the mtt assay, the agreement between the pec <dig> values obtained using both methods can be calculated, using the so-called bland-altman plot  <cit> .
for a comparison between the mtt assay and the srb assay such a bland-altman plot was reported earlier  <cit> , showing that the results obtained with the srb assay displayed a good agreement with the mtt assay results.
by plotting the difference between the pec <dig> values determined by each of the methods against the mean of the two determinations it is possible to determine the agreement between both methods  <cit> .
therefore, to compare the two methods, a bland-altman analysis of the pec <dig> values, obtained using the mtt assay and the cloneselect™ imager system, has been performed.
for example, the bland-altman plot of cisplatin after  <dig> h of incubation in both the a <dig>  and the a2780r cells  shows that there is a good agreement between the two variables, where all the data points are within the limits of agreement.
the limits of agreement are also calculated to give a quantitative analysis of the differences between both methods after  <dig> h and  <dig> h of incubation with cisplatin and oxaliplatin .
these results demonstrate that the pec <dig> values obtained with the cloneselect™ imager system and the mtt assay are in close agreement.
these observations clearly show that the cloneselect imager system is a very good alternative to the colorimetric assay to determine the antiproliferative activity of different drugs.
in order to estimate the cytotoxic activity of the platinum compounds measured with the cloneselect imager, the data analysis using the non-treated cells as 100% viable cells at the end of the assay was performed.
from independent measurements preformed using mtt and csi it is noted that the mtt assay shows larger intra-experimental deviations than the cloneselect imager system .
for instance, the dose-respond plots for cisplatin incubation after  <dig> h in the a2780r cells  show larger deviations within the data using the mtt assay than when using the cloneselect imager system .
the reproducibility is quite good when csi is used, as shown by the small errors bars in the figure.
however, larger deviations are observed for the mtt assay, as the number of viable cells increases and could account for often observed the poor linearity of the mtt assay with the cell number at high cell densities  <cit> .
similar results have been observed when the a <dig> cells are incubated with cisplatin .
cytotoxic activity in mice leukemia cells using cloneselect™ imager system
when using the mtt assay with non-adherent cells the mtt assay displays a huge disadvantage, since the culture medium has to be removed to quantify the formazan crystals at the end of the assay.
consequently, the last step in the protocol for the mtt assay results in the loss of crystals, and therefore, in large errors.
an improved colorimetric assay for non-adherent cells was introduced  <cit> , using the substrate 5--2-3- tetrazolium , where the formazan product is soluble in the cell culture medium; however, unreliable results from the mts assay in the l <dig> cell line have been reported  <cit> .
as a result of the difficulties of measuring cell cytotoxicity in non-adherent cells the cloneselect imager system was tested to determine if it was advantageous in this case.
this study was undertaken using the known cytotoxic activity of cisplatin and oxaliplatin in the l1210/ <dig> and the resistant counterpart l1210/ <dig> cells.
these cell lines are generally slightly attached to the bottom of the well, and careful handling of the microtiter plate allows accurate imaging with the cloneselect imager.
the results for the different incubation times are summarized in table  <dig>
*rf = resistance factor /ec50
as expected, oxaliplatin displays a high cytotoxic activity against the l1210/ <dig> cells  <cit> , and it is able to overcome the resistance to cisplatin present in the l1210/ <dig> cells, with no significant differences  between the pec <dig> values in the l1210/ <dig> and in the l1210/ <dig> cells for both incubation times.
the mtt assay using the mice leukemia cells could not be performed in this case, because the last step in the mtt assay requires the discharge of the medium, generating experimental errors due to the loss of formazan crystals from the non-adherent cells, as mentioned above.
this further illustrates the superiority of the cloneselect imager method.
CONCLUSIONS
the most commonly used approach to quantify mammalian cell proliferation is the conversion of a tetrazolium salt  to the formazan crystals by cellular dehydrogenases  <cit> .
nevertheless, the mtt assay is time consuming when many samples have to be assayed.
moreover, the colorimetric assay kits are relatively expensive, and most important, they are destructive, not allowing measurements over time, using the same microtiter plate.
new imaging methods have been introduced more recently, such as cloneselect™ imager.
in this study this new system is compared with the widely used mtt assay, because - despite its disadvantages - the mtt assay is largely used in the screening of the antiproliferative effect of new compounds.
a major advantage of the cloneselect imager system, compared to mtt assay, is that allows a direct screening of the antiproliferative activity of the drugs in only three minutes, with a direct overview from the cell confluence  after imaging in each well, not requiring additional steps to obtain these preliminary data.
additionally, it is possible to image the same plate as a function of time, based on a non-invasive methodology, saving in this way time and costs.
in summary, the cloneselect™ imager system has been compared with the mtt assay, and it displays lower intra experimental deviations in the dose response curves, with high reproducibility, and showing that the cloneselect imager system is a much preferred alternative to determine the antiproliferative activity of different drugs and possibly more accurate.

 high-throughput screening methods for toxicology evaluation in the early phases of drug discovery are highly desired. in the early 1980´s a novel assay for cell survival determination was reported, which has been frequently used to study the biological activity of a variety of potential cytostatic drugs. the assay was presented as a rapid, precise and simple method to detect living cells in mammalian cell cultures, using the tetrazolium salt mtt - -diphenyl tetrazolium bromide) and a microtiter plate reader. the mtt is reduced in living cells, forming formazan crystals which are then dissolved in a mixture of hcl and isopropanol, or alternatively in dimethylsulfoxide. the optical density of dissolved crystals is then read in each well within the first hour. cell survival is determined after the data analysis. although widely used, the mtt assay has several disadvantages: in particular poor linearity with the cell number, sensitivity to environmental conditions and more importantly the dependence of cell metabolism on formazan. these observations have lead to a search for different alternative assays, such as the measurement of cellular protein content with the srb dye and a variety of other in vitro assays. however, the srb assay requires a time-consuming workout, such as incubation with the dye for min and fixation. a faster and more reliable system able to screen large number of potential compounds in a large panel of cells would therefore be considered an enormous improvement in the pharmacology research. the cloneselect™ imager could offer such an opportunity. the system was designed to be capable of screening cellular confluence and growth in a range of microwell plates. for these measurements, the first step consists on a calibration of the cell number in the microtiter plates in used, and for each cell type. after this step, the cells will be counted based on such calibration. cell growth is viewed and tracked in every well, using label-free white light imaging for consistent determination of cell confluence and cell number estimation. therefore, from such measurements the confluence in each well can be determined rapidly and efficiently. the cloneselect imager system captures images of the entire well, which provides a label-free, non-destructive and direct analysis of the cell proliferation in each well as function of time. moreover, the imaging of a well plate is extremely fast, with the data acquisition in less than min. data can be either output into a spread sheet, or displayed visually as a series of pie charts. the present study attempts to validate the cloneselect™ imager system for use in cellular toxicity screening of platinum-based drugs. two different drugs have been selected for this study, namely the well-known cisplatin and oxaliplatin in two different human cancer cell lines. a comparison is made between the mtt assay results and the results obtained using the cloneselect imager system using a bland-altman plot. our results unequivocally show that the cloneselect™ imager system offers a superior and rapid alternative in determining the cytotoxicity of different agents on both adherent and non-adherent cell lines.  plates were treated with the antiproliferative agents, imaged with the cloneselect imager system h, h and h after the addition of the agents. to compare with the mtt assay, once the plate was imaged with the cloneselect imager system, the mtt assay was performed; therefore, the results from the cloneselect™ imager and the mtt assay originate from the same plate and therefore are directly comparable. the well-known human ovarian carcinoma cisplatin-sensitive a cells and the cisplatin-resistant counterpart a2780r cells were selected as adherent cells. the cytotoxic activity of cisplatin and oxaliplatin has been investigated in parallel cultures under the same conditions in a pair of human ovarian carcinoma cell lines. the incubation with the platinum compounds can be followed using the cloneselect imager system, obtaining a rapid view of the antiproliferative activity of the compounds in the cells over the time. the cell confluence in the wells versus time of incubation with the cisplatin in the a2780r cells is shown in figure the compound was added to the cells in different concentrations to achieve a dose-response curve. the a2780r cells were still growing h after the addition of the compounds; therefore, the determination of the ec value was not performed in this case. this observation is important, since the ec value after h of incubation with cisplatin in the a cell line has been reported. the same plate after h of incubation with the platinum compound shows significant changes. the cells incubated with higher concentrations show a lack of growth, while the cells incubated with lower concentrations show a growth comparable to the non-treated cells. the growth inhibition of the cells is even more evident in the wells with highest concentration of the platinum compound when the same plate is imaged after h of incubation, while the growth continue in the wells with lower concentrations of the antiproliferative agent. similar results were obtained for cisplatin incubation in the a cells, as also observed for the incubation with oxaliplatin. cell confluence is also displayed directly with the cloneselect imager system, as depicted in figure where the two first columns of the microtiter plate were non-treated cells. in addition, the plate was divided to incubate the a2780r cells with both cisplatin and oxaliplatin, as described in the experimental section, using ten different concentrations. figure clearly shows that the cloneselect imager system provides a rapid insight into the different cytotoxic activities displayed by both platinum drugs against the cells under study. the a2780r cells were treated with the same concentrations of cisplatin and oxaliplatin, confirming that oxaliplatin is more cytotoxic than cisplatin in the a2780r cells. similar results as for the cytotoxicity of oxaliplatin in the a2780r cells were observed in the a cells. moreover, it is important to mention that a different sensitivity of the a2780r cells against cisplatin was observed, compared to the sensitivity of the a cells. for instance, in figure the non-treated a2780r cells have a 55% confluence, while a 33% of confluence is observed when the cells are incubated with μm of cisplatin. in the case of a cells the sensitivity to the drug is higher, finding a 15% of confluence after h of incubation with μm cisplatin. comparison of cytotoxicity determined by cloneselect™ imager system and the mtt assay to compare the cytotoxicity results observed with the cloneselect™ imager system and the mtt assay, the agreement between the pec values obtained using both methods can be calculated, using the so-called bland-altman plot. for a comparison between the mtt assay and the srb assay such a bland-altman plot was reported earlier, showing that the results obtained with the srb assay displayed a good agreement with the mtt assay results. by plotting the difference between the pec values determined by each of the methods against the mean of the two determinations it is possible to determine the agreement between both methods. therefore, to compare the two methods, a bland-altman analysis of the pec values, obtained using the mtt assay and the cloneselect™ imager system, has been performed. for example, the bland-altman plot of cisplatin after h of incubation in both the a and the a2780r cells shows that there is a good agreement between the two variables, where all the data points are within the limits of agreement. the limits of agreement are also calculated to give a quantitative analysis of the differences between both methods after h and h of incubation with cisplatin and oxaliplatin. these results demonstrate that the pec values obtained with the cloneselect™ imager system and the mtt assay are in close agreement. these observations clearly show that the cloneselect imager system is a very good alternative to the colorimetric assay to determine the antiproliferative activity of different drugs. in order to estimate the cytotoxic activity of the platinum compounds measured with the cloneselect imager, the data analysis using the non-treated cells as 100% viable cells at the end of the assay was performed. from independent measurements preformed using mtt and csi it is noted that the mtt assay shows larger intra-experimental deviations than the cloneselect imager system. for instance, the dose-respond plots for cisplatin incubation after h in the a2780r cells show larger deviations within the data using the mtt assay than when using the cloneselect imager system. the reproducibility is quite good when csi is used, as shown by the small errors bars in the figure. however, larger deviations are observed for the mtt assay, as the number of viable cells increases and could account for often observed the poor linearity of the mtt assay with the cell number at high cell densities. similar results have been observed when the a cells are incubated with cisplatin. cytotoxic activity in mice leukemia cells using cloneselect™ imager system when using the mtt assay with non-adherent cells the mtt assay displays a huge disadvantage, since the culture medium has to be removed to quantify the formazan crystals at the end of the assay. consequently, the last step in the protocol for the mtt assay results in the loss of crystals, and therefore, in large errors. an improved colorimetric assay for non-adherent cells was introduced, using the substrate 5--2-3- tetrazolium, where the formazan product is soluble in the cell culture medium; however, unreliable results from the mts assay in the l cell line have been reported. as a result of the difficulties of measuring cell cytotoxicity in non-adherent cells the cloneselect imager system was tested to determine if it was advantageous in this case. this study was undertaken using the known cytotoxic activity of cisplatin and oxaliplatin in the l1210/ and the resistant counterpart l1210/ cells. these cell lines are generally slightly attached to the bottom of the well, and careful handling of the microtiter plate allows accurate imaging with the cloneselect imager. the results for the different incubation times are summarized in table *rf = resistance factor /ec50 as expected, oxaliplatin displays a high cytotoxic activity against the l1210/ cells, and it is able to overcome the resistance to cisplatin present in the l1210/ cells, with no significant differences between the pec values in the l1210/ and in the l1210/ cells for both incubation times. the mtt assay using the mice leukemia cells could not be performed in this case, because the last step in the mtt assay requires the discharge of the medium, generating experimental errors due to the loss of formazan crystals from the non-adherent cells, as mentioned above. this further illustrates the superiority of the cloneselect imager method.  the most commonly used approach to quantify mammalian cell proliferation is the conversion of a tetrazolium salt to the formazan crystals by cellular dehydrogenases. nevertheless, the mtt assay is time consuming when many samples have to be assayed. moreover, the colorimetric assay kits are relatively expensive, and most important, they are destructive, not allowing measurements over time, using the same microtiter plate. new imaging methods have been introduced more recently, such as cloneselect™ imager. in this study this new system is compared with the widely used mtt assay, because - despite its disadvantages - the mtt assay is largely used in the screening of the antiproliferative effect of new compounds. a major advantage of the cloneselect imager system, compared to mtt assay, is that allows a direct screening of the antiproliferative activity of the drugs in only three minutes, with a direct overview from the cell confluence after imaging in each well, not requiring additional steps to obtain these preliminary data. additionally, it is possible to image the same plate as a function of time, based on a non-invasive methodology, saving in this way time and costs. in summary, the cloneselect™ imager system has been compared with the mtt assay, and it displays lower intra experimental deviations in the dose response curves, with high reproducibility, and showing that the cloneselect imager system is a much preferred alternative to determine the antiproliferative activity of different drugs and possibly more accurate. 
traditionally cell proliferation is measured with the colorimetric mtt assay. however, the image-based technique was found non-invasive, considerably quicker and more accurate than the mtt assay. this new image-based technique has the potential to replace the cumbersome mtt assay when fast, unbiased and high-throughput cytotoxicity assays are requested. here we show that both the cloneselect imager and the mtt approach result in comparable ec values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. the cloneselect™ imager system is an image-based visualisation system for cell growth assessment.
 the cloneselect™ imager system is an image-based visualisation system for cell growth assessment. traditionally cell proliferation is measured with the colorimetric mtt assay.  here we show that both the cloneselect imager and the mtt approach result in comparable ec values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. however, the image-based technique was found non-invasive, considerably quicker and more accurate than the mtt assay.  this new image-based technique has the potential to replace the cumbersome mtt assay when fast, unbiased and high-throughput cytotoxicity assays are requested. 