MICROCAT: A Novel Cell Proliferation and Cytotoxicity Assay Based on WST-1

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1 MICROCAT: A Novel Cell Proliferation and Cytotoxicity Assay Based on WST-1 ANN-MICHELE FRANCOEUR, Ph. D. and ALFRED ASSALIAN, M.D., F.R.C.S.C. Department of Ophthalmology, Hôpital Notre-Dame Montréal, Québec, Canada H2L 4M @Compuserve.com Abstract The MICROCAT (96-well microtest plate Consecutive Assay Testing) research method was developed to accurately measure drug effects in vitro with small numbers of cells ( cells/culture). Standardization of MICROCAT using rabbit surgically explanted primary eye cells (Tenon s capsule fibroblasts, RTCF) is described. Results are shown for TCF treated with mitomycin C, rapamycin, and c-myc antisense oligonucleotides. The principles of non-destructive testing were employed; a series of three viable procedures were performed sequentially and repeatedly (i.e., daily) on the same microculture without harming the cells. The first test performed was the WST-1 colorimetric test, which measures cell metabolism (dehydrogenase activities). This was followed by Trypan Blue dye exclusion test for cell viability (cell membrane activity) and then by photographic recording to obtain information on cell number, size, and morphology. An optional final procedure was histological staining with a modified Wright s Stain prior to photographic recording. MICROCAT improved the accuracy of our experimental results by minimizing the effect of inter-culture variations, which might otherwise mask important drug effects. Other advantages include efficient use of sample material, reduced drug testing costs, relatively rapid results, and increased validity (through the use of normal cells derived from relevant tissue). Methods Cell culture Primary RTCF were surgically explanted and cultured in monolayers in 96-well flat-bottom microtest plates (Falcon #3072, Becton Dickinson), and used in passage 2 6 as previously described (1). Cells were grown in 200 µl/culture media (Dulbecco s Modified Eagle Medium [DMEM] supplemented with 15% fetal calf serum [FCS], 2 mm glutamine, 100 µl/ml penicillin, and 100 µg/ml streptomycin). Cells were counted and their viability assessed using Trypan Blue staining and hemocytometers (see below). Changing cell culture media was effected by vacuum aspiration, taking care not to allow the cultures to dry out. Multichannel pipettes and reagent reservoirs were used throughout MICROCAT, avoiding the introduction of air bubbles. Drugs Mitomycin C (MMC; Bristol-Myers Squibb Canada, Inc.), rapamycin (RAPA; ref. 1), both at 100 ng/ml, and c-myc antisense phosphorothioate oligonucleotides (AS; 20 µm, AAC GTT GAG GGG CAT, prepared by Laboratoire de Cardiologie Moléculaire, Hôpital Notre-Dame) were diluted in media, and the cultures were incubated with these reagents for the indicated times. 19

2 WST-1 assay The WST-1 assay from Boehringer Mannheim was performed according to the manufacturer s specifications. It is a colorimetric assay based on the cleavage of WST-1 tetrazolium salt by mitochondrial dehydrogenases (part of the respiratory chain) and is a measure of cellular respiration and metabolic rate. WST-1 reagent is light sensitive, and cell metabolism is temperature sensitive, so care was taken to reduce light exposure and maintain 37 C throughout. The yellow-orange colored product, a stable soluble formazan, was quantified at 450 nm using an ELISA (microtest) plate reader (model LP400, Diagnostics Pasteur, Paris). This instrument facilitates MICROCAT by simultaneously measuring absorbance of all 96 microtest plate wells, thus allowing maintenance of cell culture temperature, ph, and sterility. Background absorbance was determined by reading absorbance of cultures (cells + media) at the start of the WST-1 assay prior to reagent addition. For purposes of standardization, cultures received either 1, 10, or 20 µl of stock WST-1 reagent in 100 µl of fresh media (37 C). Cells were incubated for 1 4 hours at 37 C with duplicate hourly absorbance measurements. Based on these results, routine WST-1 assays were performed for 1 hour with 10 µl reagent. Cell viability assay The classic Trypan Blue dye exclusion staining method was employed. Cell culture medium was removed after the WST-1 assay by vacuum aspiration and replaced with 50 µl of a 1:10 (v/v) mixture of 0.4% sterile-filtered Trypan Blue Stain in Hanks Buffered Salt Solution (HBSS). After a 2 minute incubation at room temperature, the staining solution was removed by aspiration and replaced with 200 µl medium. The cultures were viewed in an inverted microscope, and the blue (dead) cells counted by eye under 40X magnification. Alternatively, the results were photographically recorded (see below). Viability was expressed as percentage unstained cells/ culture and required accurate total cell count information. Photographic recording and data analysis Cultures stained with Trypan Blue stain or with Wright s Stain (see below) were photographed with a Nikon inverted photomicroscope or with a Polaroid Instant MicroCam camera. Direct measurements of cell number, size, and morphology were obtained from prints, slides, negatives, photocopies, enlarged prints, or digitized images thereof. Results were expressed as percentage of control cultures or percentage inhibition relative to controls. Modified Wright s Stain Cultures were fixed for 2 minutes with 100 µl of a cold ( 20 C) 1:1 acetone: methanol mixture and air dried for 30 minutes to overnight. Staining was performed with 50 µl of Wright s Stain (diagnostic type R3375; BDH, Inc., Toronto) for 20 minutes and was followed by the addition of 50 µl of Wright s ph 6.4 buffer (BDH) directly on top of the stain for a further 10 minutes. The cell monolayers were then washed twice by brief emersion in phosphate-buffered saline (PBS), drained, and air dried. Nuclei stained purple, and cytoplasm stained pink. Results A MICROCAT experimental flow chart is shown in Figure 1. The method provides information on various cellular parameters (cell metabolism, number, viability, size, morphology, and number of mitotic cells) by linking the new WST-1 colorimetric metabolic assay (2,3) to classical cell biology procedures (dye exclusion testing and photographic recording). The nondestructive nature of these procedures means that they can be CELL METABOLISM CELL VIABILITY CELL NUMBER SIZE, MITOSES MORPHOLOGY PLATE CELLS IN SETS (CONTROLS & TREATED) WST-1 TRYPAN BLUE VIABILITY STAIN WRIGHT'S STAIN repeated in any order over experimental times ranging from days to months. Experiments may be terminated, and cultures histologically stained with any suitable method, such as the modified Wright s Stain described here. The basic cell culture testing unit of MICROCAT is shown in Figure 2 and consists of replicate sets of cultures containing a range of cell concentrations. Standardization of MICROCAT for a given cell type (Figure 3) involves 4 steps. Step 1, determining plating efficiency, is needed for planning experimental time courses, and this step is performed for each type of cell to be studied. In contrast, Steps 2 4 involve calibration of different assays, and a relevant cell type (e.g., rabbit TCF) is appropriate for this purpose. Typical results for standardization of MICROCAT with RTCF cells are shown in Figure 4 and Tables 1 and 2 (see also Materials and Methods ). In preliminary experiments, growth curves were performed with cultures seeded with varying numbers of cells ( cells/well). The WST-1 assay was performed every other day with different amounts of the WST-1 reagent and with incubations from 1 4 hours. Plating efficiency was poor in cultures seeded with less than 500 cells (Figure 2). Maximum absorbances in a 1 hour WST-1 assay were observed with confluent healthy cultures after 6 9 days in culture and ranged from OD 450 = 0.5 to OD 450 = 2.8, depending on the amount of WST-1 reagent used (Table 1A). Based on these results, we chose the conditions for ELISA PLATE READER MICROSCOPE ABSORBANCE DATA PHOTGRAPHIC RECORDS DIGITAL IMAGE DATA ANALYSIS Figure 1 MICROCAT flow chart. Schematic diagram of three consecutive assays performed in MICROCAT (WST-1, Trypan Blue Stain, Wright s Stain). To the left is indicated the cell biology information obtained from the assays, while to the right are listed the equipment required, the format of the results, and how they may be processed and analyzed manually with an ELISA plate reader and a standard photomicroscope (or by eye). Alternatively, microscopic images may be recorded with a digital camera and viewed directly via computer monitor. 20

3 Replicate Plate identification High-concentration drug controls Figure 2 MICROCAT experimental unit. Schematic diagram of one set of microcultures and photograph of a 96-well microtest plate with two sets of microcultures taken 15 minutes after a 2 hour WST-1 assay. The sets shown here are from Day 7 of an experiment in which RTCF cultures in Panel A received rapamycin (100 ng/ml) and those in Panel B received mitomycin C (100 ng/ml) for 2 days, and then were allowed to recover for 5 days without drug treatment. Included in this assay were high-concentration drug controls, which helped to verify that the drugs were active. As can be seen, the healthiest cultures are readily detected by eye because of their bright orange color, while cultures with poor plating efficiency (wells 1-4) or which experienced cytotoxicity due to high mitomycin C levels (wells 5-7) can be easily detected by eye and are pink in color. STEP 1 DETERMINE CELL TYPE PLATING EFFICIENCY STEP 2 STEP 3 STEP 4 CALIBRATE WST-1 CALIBRATE TRYPAN BLUE STAINING CALIBRATE WRIGHT'S STAIN A) ELISA PLATE READER 1 -FILTERS 2 -ACCURACY 3 -PRECISION 4 -BACKGROUND B) STATISTICS 1 -METHOD 2 -NO. OF REPLICATE CULTURES REQUIRED 1 SET OF MICROCULTURES Cultures (4) DRUG A-Treated Cultures C) PHOTO-MICROSCOPE % CONTROL 20% 15% 10% MICROCAT RESULTS Metab. Cell No. Viability Mitosis Cell Size Figure 3 MICROCAT calibration. The 4 steps of MICROCAT are illustrated together with idealized results comparing metabolism, cell number, viability, mitosis, and cell size of treated cultures (Drugs A-C) vs. Controls. Steps 2-3 can be performed with a standard cell type (such as rabbit TCF) and thus monitored for quality control. Step 1, determining plating efficiency, varies with different cell types (e.g., rabbit TCF cells from high vs. low passage numbers) and influences experimental design. 5% % Number of cells plated Post WST-1 Assay Drug B-Treated Cultures CONTROL DRUG A DRUG B DRUG C 96-Well Microtest Plate routine MICROCAT WST-1 assays as 10 µl of WST-1 reagent and 1 hour incubation time (conditions recommended by the manufacturer, confirming that they may be generally applicable for different cell types). The routine MICROCAT WST-1 test on subconfluent cultures typically yielded maximum absorbances around 1.5 to 1.8 OD units before subtraction of background absorbance (Tables 1B and 3 and Figure 4D). This allowed us to detect drug stimulatory effects as well as inhibitory effects, as the ELISA plate reader was capable of absorbances as high as 3 OD units at 450 nm (Figures 4A and 4B). Alternatively, 5 µl of WST-1 reagent was used in routine assays when confluent cultures were employed. Background values (culture dish plastic + media + cells + drugs or media additives) for MICROCAT were determined from absorbance readings taken at 450 nm just prior to the addition of WST-1 reagent and then subtracted from experimental values. Backgrounds were similar in the presence or absence of the WST-1 reagent and ranged from 0.16 to 0.21 OD units in cultures with low vs. high cell densities (Table 1B and Figure 4D). Using other filters as a reference filter was found to be less accurate, as absorbance tended to be lower by OD units (Table 1D). This would lead to an underestimation of druginduced inhibition of cell metabolism, especially in sparse cultures. Phenol red indicator dye absorbs about 0.1 OD unit/100 µl volume media at 450 nm (Table 2); thus, sensitivity may be improved by omitting the dye from the culture media. In our hands, RTCF needed 2 3 days in culture to establish themselves, and conditions of low plating efficiency could readily be detected by eye (see for example, Figure 2, Panel A) due to the difference in color of healthy (orange) vs. unhealthy (pink) cultures after the WST-1 assay. To avoid complicating experiments by poor plating efficiency, RTCF were routinely plated three days prior (Day -3) to addition of a drug on Day 0, and time points were typically taken at Day 0, 1, 3, and 7 (Figure 4D). The WST-1 assay sensitivity limit for RTCF cells was about 100 cells/well, which gave an absorbance of 0.05 OD units greater than background. 21

4 3,2 2,8 2,4 2,0 0 A B C D 10 1,1, ul WST-1 Product ul WST-1 Product 3,2 2,8 2,4 2,0 Plate Med 100 ul Med 200 ul W 50 ul E1 W 50 ul E2 W 100 ul E1 W 100 ul E2 W150 ul E1 W150 ul E2 W 200 ul E1 W 250 ul E1 W 300 ul E1 1,8 1,4 1,0 0,6 0,2-1 0 CONTROL CULTURES Days Post Drugs BGD 7 Figure 4 WST-1 assay calibration. Panel A. Standard curve relating absorbance at 450 nm to increasing amounts of culture media post WST-1 assay (containing orange-colored product). This type of curve has various uses, such as (i) facilitating experimental design (e.g., determining what culture media volume to use when relating one cell type to another or when scaling up from one culture plate size to another) or (ii) for calibration of pipettes or ELISA plate readers or filters (see Table 2). Panel B. Same standard curve data as in Panel A, depicted with a logarithmic Y axis and with error bars omitted for clarity, to illustrate the high sensitivity of the ELISA plate reader. Panel C. Comparison of various sources of experimental error in the WST-1 assay. Absorbance at 450 nm and experimental error (based on 4 replicates) of microtest plate plastic (Plate), different volumes of media (Med), duplicate (E1 and E2) ELISA plate reader data for the same WST-1 product volumes (W). Panel D. Representative WST-1 assay results for an experiment with RTCF (passage 6) where sets of microcultures were plated with cells/culture (4 replicates) at Day 3, and WST-1 assay time points were taken at Days 0, 1, 3, and 7. Media was changed on Days 0 and 4. For clarity, the data is shown without error bars, which varied from ±3 22%. Background (BGD) was subtracted from experimental data and is also shown separately for comparison. SD, standard deviation; SE, standard error; CV, coefficient of variation. Sources of WST-1 experimental variability are listed in Table 2. Media containing the orange colored product (OD >1.2) was collected post WST-1 assay, pooled, frozen, and subsequently used to calibrate instruments such as the ELISA plate reader (see Figure 4C), filters (Table 1C), and multichannel pipettes (Table 2). While absorbance was proportional to volume of WST-1 (Figure 4A), the precision of the plate reader varied from a low of 2% to a high of about 8%, with maximum imprecision between OD units (Figures 4A, 4C, and Table 2). Thus, the experimental variations introduced by the ELISA plate reader were sometimes comparable to inherent inter-culture variations (5 40%) seen with primary cultures (cell strains); see Figure 6 and Table 3 for examples. Referring to Tables 1 3, by far the largest sources of experimental variations were the interculture variations, followed by the ELISA plate reader and multichannel pipettes. An avoidable large source of experimental error was due to air bubbles present during the ELISA plate absorbance measurements, which could increase absorbance by 0.8 OD units. We developed procedures to detect and rule out outliers (defined as data greater than 3 standard deviations from the mean) in our data and to carefully track errors in all experimental manipulations. Two procedures improved our ability to detect outliers: (i) We continued the WST-1 incubation for a second hour and compared the hour 2 data to the hour 1 data when an outlier was suspected. (ii) Duplicate ELISA plate readings, about 1 minute apart, were also taken at each experimental time point and after each hour of incubation (see Figure 4C). Both procedures are now routinely performed and have helped to trace the source of the variation to inherent inter-culture variation, variation introduced by the ELISA plate reader, human error, or other equipment failure (e.g., printer or pipetting error). For microcultures with high absorbance values in the WST-1 assay (OD >3.0), 10 µl and 50 µl aliquots were removed and the absorbance reread at 450 nm. Results were then corrected for volume using a standard curve (Figure 4A). This method is recommended by Boehringer Mannheim and proved more accurate than dilution of samples (data not shown). Alternatively, cultures with high absorbances could be reread with other filters (wavelengths of 405 nm or 490 nm), and the results could be multiplied by the empirically derived absorbance correction factor constants indicated in Table 1C. Calibration of MICROCAT step 3 was straightforward. Trypan Blue staining was performed as described in Materials and Methods. Reversing the order of the WST-1 and Trypan Blue tests did not significantly affect the results of either test (data not shown), at least for the short incubation times used. Viability was reduced in cultures that had been exposed to either reagent for 4 hours (< 90% viability) or overnight (< 50% viability). MICROCAT step 4, the calibration of Wright s Stain, was similarly straightforward (see Materials and Methods and Figure 5). MICROCAT offers the possibility of research testing drug effects on small numbers of primary cells in vitro (Figure 6). RTCF cultures were plated (Day -3), grown for 3 days, and then treated with mitomycin C (100 ng/ml), rapamycin (100 ng/ml), or c-myc anti-senseoligonucleotides (20 µm) for 2 days, and then allowed to recover for 5 days. Figure 6 shows WST-1 data for the Day 3 time point (1250 cells plated on Day -3). The statistical analysis of this data is provided in Table 3, while a summary of the MICROCAT experiment is presented in Figure 7 (error bars omitted for the sake of clarity). Figure 5 displays representative photographic records used to count cells, measure cell size, and assess 22

5 A C B D morphology of control and drug-treated cultures for Day 7 of this experiment. All three types of drugs produced a similar 50 60% inhibition of cell metabolism and blocked mitosis with relatively little effect on cell viability (Figure 7). The effects on cell number and size were more complex and in some cases (c-myc antisense or rapamycin) were reversible. While mitomycin C (Figure 5, Panel B) caused the cells to grow very large (2.5 times the area of control cells, Panel A), rapamycin had little effect on cell size (Panel C), and c-myc antisense-treated samples contained very many tiny cells (Panel D). These results extend our previous results with rapamycin (1) and support observations made by other groups using RTCF cells and mitomycin C (4 6). A detailed report of our work with MICRO- CAT and human and rabbit TCF are beyond the scope of this article and will be presented elsewhere (Francoeur and Assalian, manuscript in preparation). Figure 5 Photographic records of MICROCAT assays. Shown are Wright s stained cultures on Day 7 (5 days recovery after 2 days of treatment with drugs) from the same experiment described in the text and shown in Figure 6. Panel A: Control cultures Panel B: Cultures treated with 100 ng/ml mitomycin C Panel C: Cultures treated with 100 ng/ml rapamycin Panel D: Cultures treated with 20 µm c-myc antisense oligonucleotides. Discussion MICROCAT is being used at the research level to study the effects of various drugs currently used as glaucoma adjuvant therapy (e.g., mitomycin C, 5-fluorouracil) and potentially useful drugs (rapamycin and c-myc antisense 1,8 1,4 1,0 0,6 0,2 WST-1, RTCF, Day 3 (1250 Cells plated on Day -3) CONTROL AS20 RAPA MITC BGD TREATMENT Figure 6 Histogram showing data from a representative MICROCAT WST-1 experiment with RTCF cells. Cells were plated with 1250 cells/well on Day -3, treated with the indicated drugs for 2 days (Day 0-Day 2), and allowed to recover for 5 days as described in the text. (See Table 3 for statistics). AS20 = 20 µm c-myc antisense oligonucleotides; RAPA = 100 ng/ml rapamycin; MITC = 100 ng/ml mitomycin C. % OF CONTROL VALUES 12% 10% 8% 6% 4% 2% % MICROCAT SUMMARY - DAY 3 (1500 Cells plated Day -3) Metab. Cell No. Viability Mitosis CONTROL AS20 RAPA MITC Figure 7 Summary of MICROCAT DATA for Day 3 of the experiment described in the text and in part in Figures 5 and 6. The results were expressed as percentage of control culture values for purposes of comparison. 23

6 Volume of WST-1 (µl) Maximum OD A) Standardization of WST-1 assay on RTCF cells. 4 replicates, 1 hr 37 C, 100 µl media; 6 days in culture, 4000 cells plated on Day 0. OD 450 Coefficient Standard Standard of variation deviation error Routine assay, maximum mean Day 6 in culture (4) absorbance observed = ± (13.7%) at any time point Mean background absorbance Day 6 in culture WST-1 reagent (cells µl ± (7.1%) media + plastic); n = 16 Mean background absorbance Day 1 (min) Day WST-1 reagent (cells µl (max) in culture media + plastic); n = 16 OD = ± (7.4%) B) Routine WST-1 assay on RTCF cells. Absorbances at 450 nm were measured on confluent cultures ( cells plated Day -3); 100 µl media, 98% viable cells. 450 nm 405 nm 490 nm Absorbance correction factors x 1 (none) x 1.25 x 3.22 (OD < 0.25) x 2.63 (OD = ) C) Comparison of ELISA plate filters in WST-1 assay with RTCF cells. 450 nm 630 nm Mean of 18 determinations ± 4% ± 4% (cells plus 100 µl medium plus plastic) D) Comparison of backgrounds in WST-1 assays. Day 3 measurements, 1500 RTCF cells plated; 10 µl, 1 hr. Table 1. Various measurements taken during standardization of WST-1 assay for MICROCAT. WST-1 Assay: Sources of variation that influence Absorbance at 450 nm experimental error. Differences between different ELISA plate readers OD units measuring the same sample (comparison of 96 samples) ± 5% 15% (depends on OD) Differences between different channels of a OD units multichannel pipette (8 or 12) ± 5 13% (depends on OD) (calibration of 2 different pipettes) Differences between means of 2 consecutive sets << 0.05 OD units, < 1% of ELISA plate readings of a set of 8 samples of mean OD = (3.8%) 100 µl WST-1 product of absorbance (both determinations gave the same mean) OD = 1.00 (same machine) Differences between absorbance of plastic of different << 0.05 OD units, < 1% wells on microtest plate and between 2 different mean OD = ± (8.8%) microtest plates (96 determinations on each plate) (for 1 or 2 two plates) Differences between background absorbance of < 0.05 OD units cultures (plastic + cells µl media) with ± (8.2%) (low) different cell densities. Comparison between high ± (15.3%) (high) (Day 6) and low (Day 1) RTCF cultures. Difference OD = (8.8%) Absorbance due to 100 µl media alone (includes plate << 0.05 OD units but no cells, mean of 18 different determinations, ± (6.7%) same pipette used for each). No statistically significant difference observed with or without WST-1 reagent. Differences due to presence of small or large air bubbles in 100 µl media in wells of microtest plate, (can be much greater than sample absorbance) includes plastic but no cells oligonucleotides) in vitro, with small numbers of patients early passage TCF cells. Antisense oligonucleotides (reviewed in ref. 7), particularly those that block the expression of such key cell cycle control proteins as c-myc, offer the promise of new therapeutics in ophthalmology, with high specificity and minimal side effects. MICROCAT largely depends on the sensitivity and reproducibility of the WST-1 assay and the fact that this test is not cytocidal, at least during short (1 2 hour) assays. The Trypan Blue dye exclusion test is useful for assessing membrane integrity/ cell viability, but is not informative when cells have functioning membranes but low or no potential for proliferation (as occurred with mitomycin C). We found the Polaroid MicroCAM camera valuable in speeding up results and expect that a digital camera would facilitate experimental throughput even more. The improved accuracy and validity of our results with MICROCAT relative to other methods of drug testing/evaluation becomes more evident as the inherent inter-culture variations become large (>± 30%, CV >15). This first communication on MICRO- CAT involves manual testing; however, automation is certainly feasible, as are further miniaturization and additional types of testing (e.g., PCR-based testing). Clearly, the MICROCAT can accommodate other types of cells, as well as other drugs and variations in the sequence and type of intermediate non-destructive testing performed. A limiting factor is our inability to obtain 96-well microtest plates for tissue culture with detachable wells (as have been developed for immunological assays) or with UV-permeable wells. This would allow us to continue microculture testing with a longer series of tests using other techniques (e.g., immunochemical, molecular biological, biochemical). References 1. Salas-Prato, M., Assalian, A., Mehdi, A. Z., Duperré, J., Thompson, P. and Brazeau, P. (1996) Inhibition by rapamycin of PDGF- and bfgf-induced human Tenon fibroblast proliferation in vitro. J. Glaucoma 5: Ishiyama, M., Masanobu, S., Sasamoto, K., Mizoguchi, M. and He, P. (1993) A new sulfonated tetrazolium salt that produces a highly watersoluble formazan dye. Chem. Pharm. Bull. 41: Table 2. Sources of variation in WST-1 assay. 24

7 Statistic Control c-myc Rapamycin Mitomycin C Background antisense (100 ng/ml) (100 ng/ml) oligonucleotide (20 µm) % of control 100% 50.0% 47.6% 36.2% 0.0% % inhibition 0% 50% 52.4% 63.8% Mean background Mean Range (min-max) Range Error (13.8%) (27.2%) (30.2%) (17.2%) (4.4%) Standard deviation t-test sig. C/Dx* Significant* Yes Yes Yes Yes No Yes Yes No Yes Yes Yes Yes Yes Yes Yes Coefficient of variation Standard error Table 3. Statistical analysis of representative MICROCAT WST-1 experimental data illustrated in the histogram in Figure 6 and for the experiment described in the text. A representative MICROCAT WST-1 Day 3 time point (1250 cells plated on Day - 3) was assayed after 2 days of drug treatment (Days 0 2) and 1 day of recovery. *t-test sig. C/Dx: Comparison of Student t-test significances of WST-1 results for control cultures vs. those of drug X-treated cultures 3. Ishiyama, M., Tominaga, H., Shiga, M., Sasamoto, K., Ohkura, Y., Ueno, K. and Watanabe, M. (1995) Novel cell proliferation and cytotoxicity assays using a tetrazolium salt that produces a water-soluble formazan dye. In Vitro Toxicology 8: Yamamoto, T., Varani, J., Soong, H. K. and Lichter, P. R. (1990) Effect of 5-fluorouracil and mitomycin C on cultured rabbit subconjunctival fibroblasts. Ophthalmol. 97: Khaw, P. T., Ward, S., Porter, A., Grierson, I., Hitchings, R. A. and Rice, N. S. C. (1992) The longterm effects of 5-fluorouracil and sodium butyrate on human Tenon s capsule fibroblasts. Invest. Ophthalmol. Vis. Sci. 33: Bershadskii, A. D. and Stavrovskaia, A. A. (1991) The normalization of tumor cell morphology related to an increase in their size: research on giant cells produced by mitomycin C treatment. Tsitologiia 33:67-75 (title translated from Russian). 7. Prins, J., de Vries, E. G. E. and Mulder N. H. (1993) Antisense of oligonucleotides and the inhibition of oncogene expression. Clinical Oncology 5: Acknowledgments Support for this study was provided by the ophthalmologists of Hôpital Notre-Dame and Alcon Canada, Inc. Product Cat. No. Pack Size Cell Proliferation tests Reagent WST-1 25