previous reports, it was shown that phenol red does not interfere with the measurements and no washing steps are required since all ingredients can be added ...
Journal of Immunological Methods, 119 (1989) 203-210
203
Elsevier JIM05150
Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill M o r t e n Bagge H a n s e n , S v e n d E r i k N i e l s e n a n d K u r t Berg The Interferon Laboratory, Institute of Medical Microbiology, University of Copenhagen, Juliane Mariesvej 22, DK-2100 Copenhagen O, Denmark
(Received 11 May 1988, revised received 28 October 1988, accepted 5 December 1988)
The tetrazolium salt (MTT) method involving conversion of M T T to coloured formazan by cells serving as indirect measurements of cell growth/cell kill has been reported by several groups, although technical problems have been encountered. The present investigation was undertaken in order to delineate what laboratory variables have direct influence on the sensitivity and reproducibility of the method. The p H of the extraction buffer was of the utmost importance, since it was demonstrated that a p H > 5 would give rise to false signals. Furthermore, modifying the composition of the extraction buffer, all formazan dye grains were solubilised, totally. A direct comparison with published methods demonstrated that only the modified method would yield 100% higher signals without increasing the background. In contrast to previous reports, it was shown that phenol red does not interfere with the measurements and no washing steps are required since all ingredients can be added subsequently. Serum proteins at concentrations up to 25% have no influence on the result. All samples can be measured in an ELISA scanner at 570 nm with little intra-assay variation. Key words: Tetrazolium salt; Formazan; Cell growth; Cell kill
Introduction Originally, M o s m a n n (1983) described the general principle involved in the detection of cell growth or cell kill as indicated by the conversion of the tetrazofium salt to the coloured product, formazan, the concentration of which can be mea-
Correspondence to: K. Berg, The Interferon Laboratory, Institute of Medical Microbiology, University of Copenhagen, 22 Juliane Mariesvej, DK-2100 Copenhagen O, Denmark. Abbreviations: FCS, fetal calf serum; NBS, new born calf serum; PBS, phosphate-buffered saline; DMF, N,N-dimethyl formamide; SDS, sodium dodecyl sulphate; MTT, tetrazolium salt.
sured spectrophotometrically. At present, the formation of formazan is thought to take place via intact mitochondria, although other extracellular locations with dehydrogenase activity may contribute to the total formazan production (Mossmann, 1983). The M T T approach has been valuable for the detection in bio-assays of, for example, lymphotoxin (Green, 1984), growth factors (Denizot et al., 1986), interleukin (Heeg et al., 1985), interferons (Berg et al., 1988) although more recently several authors have alluded to technical problems giving rise to great variation (Denizot et al., 1986; Espevik et al., 1986; T a d a et al., 1986; Neville, 1987). Since we experienced similar variation at the beginning of this study, we decided to explore the
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
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critical laboratory parameters involved in the technique. Optimisation was attempted and direct comparisons made with published methods.
Materials and methods
Cell lines The Wehi 164 cell line, clone 13, mouse fibrosarcoma cell line, was provided by T. Espevik, University of Trondheim, Norway. The cells were cultured in RPMI 1640 including 15% FCS supplemented with 0.1 mM glutamine, 30 t~g/ml gentamicin, and 5% N a H C O 3 (Espevik et al., 1986). MDBK, A549 and Vero cells were grown in Eagle MEM including 5% FCS, supplemented with 1% penicillin and streptomycin, 1% L-glutamine and 5% N a H C O 3. Cells were grown in Nunc clone plastic bottles (TecNunc, Roskilde, Denmark) and split twice weekly at different cell densities according to standard procedures using trypsin. Wehi 164 cells were split without using trypsin. Daudi cells were obtained from Dr. Strander, Stockholm, and grown as a suspension culture. M D B K and Vero cells were obtained from the American Type Culture Collection. A 549 cells were obtained from Dr. A. Meager, National Lab. Biol. Control, London. Chemicals MTT(3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) was purchased from Sigma (cat. no. M2128). It was dissolved at a concentration of 5 m g / m l in sterile PBS at room temperature and the solution was further sterilised by filtration and stored at 4 ° C in a dark bottle equipped with a tight cap. It was prepared freshly each month. SDS (practical grade) was obtained from Sigma and N, N-dimethyl formamide (DMF) was purchased from Fluka (analytical grade, cat. no. 40250). Lysing buffer was prepared as follows: 20% w / v of SDS was dissolved at 3 7 ° C in a solution of 50% of each D M F and demineralised water, using magnetic stirring; p H was adjusted to 4.7 by adding 2.5% of an 80% acetic acid and 2.5% 1 N HC1. MTT/formazan extraction procedure 25 ~1 of the 5 m g / m l stock solution of M T T were added to each well, and after 2 h of incuba-
tion at 37 o C, 100/~1 of the extraction buffer were added. After an overnight incubation at 37 ° C, the optical densities at 570 nm were measured using a Titer-Tech 96-we!l multiscanner, employing the extraction buffer as the blank. Unless specified in the text, no mixing was performed at all, and no medium was removed prior to the addition of any ingredient.
Neutral red procedure The medium was removed by inversion of the tray. The cell layer was washed twice with PBS pH 7.4, and the medium replaced by 100 /~1 MEM containing 67/~g/ml of neutral red dye. After 2 h incubation at 37°C, the wells were inverted, washed twice with PBS pH 7.4, and the dye absorbed by the cells extracted with 100 #1 of 50% ethanol in 1% acetic acid. Colour development in the microtrays was then quantified at 540 nm in a TiterTech multi-scanner (Finter, 1969; Forti et al., 1986).
Results
Initial experiments showed excessive variability in the reproducibility and sensitivity of the MTT assay (Mosmann, 1983). We therefore explored what variables affected reliability by investigating the effect of M T T concentration and incubation times with MTT, the number of seeded cells, the metabolic activity of the cells, the influence on the OD signal of different methods of formazan extraction, and the influence of pH. A modified M T T system was developed and applied to a long-term culture experiment. The results were compared to those obtained using a neutral red dye uptake method (Finter, 1969).
Concentration of M T T Previously published reports (Mosmann, 1983; Heeg et al., 1985) have suggested that the concentration of MTT in the medium should be between 0.5-2.5 m g / m l . Using the standard procedure shown in Table II, we tested a variety of human, bovine, and murine cell lines and found that if the final concentration of MTT was maintained at about 1 m g / m l , the OD signal correlated in a linear fashion with the cell number (data not
205 OD 570
t w e e n t h e n u m b e r o f s e e d e d cells a n d the O D readings. This linearity was only observed within t h e r a n g e o f 3 0 0 - 5 2 000 c e l l s / w e l l . C e l l n u m b e r s b e l o w 3 0 0 / w e l l y i e l d e d a signal w h i c h w a s n o t r e p r o d u c i b l e , a n d a b o v e 50 000 t h e c u r v e r e a c h e d a plateau. Similar findings were noted employing o t h e r cell lines. T h e t i m e c o u r s e f o r i n c u b a t i o n o f t h e cells w i t h M T T w a s o p t i m i s e d . F r o m Fig. 2 it c a n b e s e e n t h a t t h e O D c u r v e s r e a c h e d a p l a t e a u a f t e r 1 h. O t h e r cell lines w e r e t e s t e d a n d y i e l d e d s i m i l a r results; it w a s t h e r e f o r e d e c i d e d to i n c u b a t e all cell c u l t u r e s f o r o n l y 2 h at 37 ° C w i t h M T T .
2.0
1.5
10
05
number of seeded cells/well
Fig. 1. The OD signal as a function of the number of cells. Wehi-164 cells were seeded in RPMI and 18% fetal calf serum, at increasing densities in 100 /~1 medium. After 20 h incubation, at 37°C, 5% CO 2 the MTT protocol (Table II) was followed. The data represent means and standard deviations of triplicates. shown). This concentration of MT'F was therefore u s e d in all o f the e x p e r i m e n t s d e s c r i b e d .
The number of seeded cells and the time course for the M T T incubation Fig. 1 s h o w s a n a l m o s t l i n e a r c o r r e l a t i o n b e -
The influence of the metabolic level I n o r d e r to i n v e s t i g a t e the r o l e o f cell m e t a b o l i s m in f o r m a z a n p r o d u c t i o n , M T T c o n v e r s i o n at different temperatures was determined. As shown in Fig. 3, t h e O D signal at r o o m t e m p e r a t u r e w a s o n l y 1 / 3 o f t h a t at 37 ° C. I n c r e a s i n g t h e i n c u b a t i o n t e m p e r a t u r e to 40 ° C y i e l d e d o n l y a m i n o r i n c r e a s e , a n d 3 7 ° C was t h e r e f o r e u s e d as t h e M T T i n c u b a t i o n t e m p e r a t u r e in this study.
The influence of different extraction procedures I n i t i a l l y the e x p e r i m e n t s s h o w e d a g r e a t d e a l o f v a r i a t i o n w h e n e x t r a c t i o n o f t h e f o r m a z a n d y e was
OD570 '
I000 m
500
c.-
500
~?~
i
2
7 /. time hours
Fig. 2. The OD signal as a function of the time of incubation with MTT. Vero cells were seeded in microwells on day 1:5000 (ix), 15000 (©), 30000 (rq) and 45000 (v) cells/well, respectively in 150/~1 medium, including 5% NBS, and incubated at 37 ° C, 5% CO 2. Medium and MTT alone (e). On day 2, 25 ~1 of MTT (5 mg/ml) were added to each well and the wells were incubated 0.5, 1, 2, and 4 h at 37°C, respectively. After the addition of 100/~1 D M F / S D S extraction buffer OD measurements at 570 nm were made - the data represent the means of triplicate samples, standard deviations ranging from 1 to 10% (not shown).
0
5000
15000
30.000 45000 number of seeded ceUs/weU Fig. 3. The influence of temperature on the OD during the MTT incubation. On day 1 microplates were seeded with Vero cells at 5000, 15000, 30000 and 45000 cells/well in 150 /~1 medium including 5% NBS and incubated overnight at 37 o C. On day 2 the MTT protocol (cf. Table II) was followed. (v) MTT incubation at 40 o C. (©) MTT incubation at 37 ° C. (zx) MTT incubation at 20 o C. The data represent the means of triplicate samples. (Standard deviations were all
