Use of a filtration plate in the MTT assay

Toxic. in Vitro Vol. 8, No. 4, pp. 739 741, 1994

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Pergamon

0887-2333(94)00117-0

Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0887-2333/94$7.00+ 0.00

USE OF A F I L T R A T I O N PLATE IN THE MTT A S S A Y D. SLADOWSKI*,S. STEER, R. CLOTHIERand M. BALLS FRAME Alternatives Laboratory, Department of Human Morphology, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK and *Department of Transplantology, Institute of Biostructure, Medical Academy Warsaw, ul. Chalubiflskiego 5, 02-004 Warsaw, Poland Abstract--The use of the MultiScreenfiltration plate enables fluids to be removed efficientlyfrom the plate without disturbing cells, or formazan crystals formed during the MTT test. Using a I : 1 mix of dimethyl sulfoxide and ethanol as solvent allows optical densities to be measured directly on the Multiscreen plate. The possibility of washing cells without losses in their number appears to be advantageous especially for in vitro studies on cytokines and on the cytotoxicity of coloured substances, such as dyes. In this study the results of cytokine assessment using the Multiscreen plate were compared with those obtained using a normal fiat-bottomed plate. Two methods of assessment of the cytotoxicity of the dye rose bengal were also compared.

INTRODUCTION

The study presented in this paper describes the use of the Multiscreen plate for cytokine assessment and for assessment of the cytotoxicity of coloured substance. Both tests were performed using the Multiscreen plate and a normal flat-bottomed plate. Rose bengal was used as the coloured substance because of its strong colour and relatively low toxicity.

The MTT assay (Mosmann, 1983) is based on the conversion of the yellow tetrazolium salt, 3-(4,5dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) to the coloured formazan product, by mitochondrial enzyme activity of viable cells. The concentration of the coloured product can be measured spectrophotometrically and equated to cell number. The assay has found widespread application for the testing of chemosensitivity (Arnould et al., 1990; Romijn et al., 1988) and radiosensitivity (Price and McMillan, 1990), the assessment of cell simulation in immunology (Niks et al., 1990) and of cytotoxicity effects (Ferrari et al., 1990), the evaluation of cytokine production (Leslie and Hay, 1991), the appraisal of anthelmintic drug sensitivity and for toxicity testing (Comley and Turner, 1990). Since the publication of this method there have been numerous modifications, but none of these is without problems. Because of the coloured product, it is extremely difficult to use the MTT assay for cytotoxicity studies of coloured substances. For obvious reasons it is not possible to add the extraction buffer directly to the medium to extract the formazan crystals. It is also very difficult to remove medium containing the tested coloured substance without aspirating the formazan product from the culture well. As a result of these difficulties the MultiScreen filtration plate has been used, enabling the tested dye to be washed out without losing any cells. Cells were grown on a filtration plate (Multiscreen HV) provided by Millipore and the assay was designed so that the entire procedure including reading the optical densities, could be performed on the same plate.

MATERIALSANDMETHODS

Abbreviations: DMSO=dimethyl sulfoxide; IL-2=inter-

leukin 2; PBS = phosphate buffered saline.

Cell lines and chemicals. The murine cytotoxic T cell line, CTLL-2, was kindly provided by Dr Ian Gibb (Boots Co. plc, Nottingham, UK). The human, interleukin 2 (IL-2)-producing, lymphoma cell line was obtained from ECACC. Cells were cultured in RPMI-1640 medium (Gibco, Paisley, UK) with 10% foetal calf serum (Gibco), 2 mM L-glutamine, 5 x 10-SM 2-mercaptoethanol (Sigma, Poole, UK), 2 # g fungizone/ml (Squibb, Hounslow, UK) and 100/lg gentamicin/ml (Gibco). Medium for CTLL-2 cells was additionally supplemented with 50 U human IL-2/ml (Lymphocult-T-HP, Biotest Diagnostics, Solihull, UK). Cells were grown in 50-ml culture flasks at concentrations ranging from 104 to 105 cells/ml and subcultured every 3 days, at 37°C in 5% CO2/95% air in a LEEC humidified incubator. Rose bengal was purchased from Sigma. M T T assay on the MultiScreen filtration plate. Cells were cultured in Multiscreen HV filtration plates (Millipore, St Quentin-Yuelines, France). The Multiscreen plates are modified 96-well plate units with a microporous Millipore membrane attached to each well. MTT was dissolved in the culture medium at a stock concentration of 5 mg/ml and used that day. A 50-/al aliquot of the MTT was added to each well of the assay plate and incubated for 4 h r at 37°C. The medium was removed by aspiration through the

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IL-2 concentration (units/ml) Fig. 1. Determination of interleukin 2 (IL-2) concentration by MTT assay, using the Multiscreen filtration plate (11) or a standard flat-bottomed plate ( ~<).

were subsequently read using the same settings as above. I L - 2 assay. CTLL-2 cells, an IL-2 responding cell line, were washed three times to remove any residual cytokines. The cells were resuspended at a cell density of 1 x 105/ml in medium without additional IL-2, and 100 #i per well was aliquoted on to the Multiscreen plate. The medium from the Multiscreen plate was aspirated through the filters, after which 200/zl preprepared concentrations of IL-2 in medium was added to each well. The plate was incubated at 37°C for 48 hr after which the M T T assay was performed. Cytotoxicity assay. The chemical to be tested was dissolved over a range of concentrations in culture medium. 100/d 5 x 104 Jurkat E.6.1 cells/ml was added to each well 12hr before adding the test chemical. 100/11 of the test chemical solution was added to each well. The test was performed in triplicate using standard fiat-bottomed plates and Multiscreen filtration plates. After 24 hr of incubation the medium was removed and cells were washed three times using 200/~1 PBS each time. The

filters using the vacuum manifold for the multiscreen by Millipore. The cells were then washed with 200/11 phosphate buffered saline (PBS) and aspirated through the filters as before. The plate was disconnected from its support and left to air-dry for several minutes. To dissolve formazan crystals and enable optical density measurements to be made directly on the filtration plate, 100 #1 of a 1 : 1 dimethyl sulfoxide ( D M S O ) and ethanol mixture was used. The plate was shaken gently for approximately 5 min before reading (measurement 540 nm; reference 620 nm) on an Anthos 2000 plate reader. M T T assay on a standard f l a t - b o t t o m e d plate. Cells were cultured on fiat-bottomed 96-well plates (Nunc, Roskilde, Denmark). The M T T stock solution was added and the incubation was performed as above. After incubation, the plates were centrifuged at 90 g for 10min, after which they were rapidly inverted with a firm flick to remove the medium. To solubilize the crystals, 150 pl of a 1:1 mix of ethanol and D M S O was added to each well and the plates were shaken for about 20 min. The plates

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mg/litre Fig. 2. Determination of the cytotoxicity of rose bengal using the Multiscreen filtration plate (11) or a standard flat-bottomed plate ( :~ ).

Use of filtration plate in MTT assay

741

M T T test was subsequently performed on both plates,

assay for in vitro studies on the cytotoxicity of coloured substances.

RESULTS

Acknowledgement--This work was supported by industrial sponsors of the FRAME Research Programme.

In both assays the use of the Multiscreen filtration plate gave better results than using the standard flat-bottomed plate. Figure 1 shows the correlation between IL-2 activity and optical density in our IL-2 assay. Both tests gave similar dose-response curves; however, higher optical densities and smaller errors were observed in the experiment performed on the filtration plate. In the experiment with rose bengal, the test performed on flat-bottomed plates failed to give any positive results. The IDs0 value obtained using the filtration plate was 180 mg/litre (Fig. 2). DISCUSSION The results revealed that when the assay is performed on a Multiscreen filtration plate, as opposed to the assay performed on the conventional flatbottomed plate, the results are dependent only on number and metabolic activity of cells and are not affected by residual cytokine or coloured substance. Where the flat-bottomed plate was used, the resultant optical densities of residual dye were higher than those obtained from formazan solubilization, thus suggesting (erroneously) that rose bengal increases the viability of cells in a dose-dependent manner. This error did not occur when the experiment was performed using the filtration plate. This could be ascribed to a reduction in the residual dye during the washing steps of the assay, compared with the conventional method. As it is possible to remove almost completely the test dye from the well, the use of a filtration plate makes it possible to use the M T T

REFERENCES

Arnould R., Dubois J., Abikhalil F., Libert A., Ghanem G., Atassi G., Hanocq M. and Lejeune F. J. (1990) Comparison of two cytotoxicity assays: tetrazolium derivative reduction (MTT) and tritiated thymidine uptake on three malignant mouse cell lines using chemotherapeutic agents and investigational drugs. Anticancer Research 10, 145-151. Comley J. C. and Turner C. H. (1990) Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability. International Journal of Parasitology 20, 251-255. Ferrari M., Fornasiero M. C. and Isetta A. M. (1990) MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. Journal of Immunological Methods 131, 165-167. Leslie H. and Hay F. C. (1989) Practical Immunology. 3rd Ed. Blackwell, Oxford. 429 pp. Mosmann T. J. (1983) Rapid colorimetrie assay for cellular growth and survival: Application to proliferation and cytotoxic assays. Journal of Immunological Methods 65, 55-57. Niks M., Otto M., Busova B. and Stefanovic J. (1990) Quantification of proliferative and suppressive responses of human T lymphocytes following ConA stimulation. Journal of Immunological Methods 126, 263-271. Price P. and McMillan T. J. (1990) Use of the tetrazolium assay in measuring the response of human tumor cells to ionizing radiation. Cancer Research 50, 1392-1996. Romijn J. C., Verkoelen C. F. and Shroeder F. H. (1988) Application of the MTT assay to human prostate cancer cells lines in vitro: establishment of test conditions and assessment of hormone stimulated growth and druginduced cytostatic and cytotoxic effects. Prostate 12, 004)9.