Journal of lmmunological Methods, 142 (1991) 257-265. © 1991 Elsevier
Science Publishers B.V. All rights reserved 0022-1759/91/$03.50. ADONIS ...
257
Journal of lmmunological Methods, 142 (1991) 257-265
© 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50 ADONIS 002217599100283C JIM06058
An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT N e a l W. R o e h m , G e o r g e H. R o d g e r s , S t e p h e n M. H a t f i e l d a n d A n d r e w L. G l a s e b r o o k Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN46285, U.S.A.
(Received 12 February 1991,revised received 20 May 1991, accepted 21 May 1991)
A new tetrazolium salt XTI', sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of X T T by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of X T T by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of X T T by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, M C / 9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with X T T than were observed with MTT. Thus the use of X T T in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity. Key words: Colorimetric assay; Proliferation assay; Tetrazolium salt; XTT
Introduction
Colorimetric assays of cell viability, activation and proliferation based on the use of the tetraCorrespondence to: N. Roehm, Building 98C/4, Lilly Corporate Center, Indianapolis, IN 46285, U.S.A. Abbreviations: CAS, concanavalin A induced supernatant; ConA, concanavalinA; IL-2, interleukin-2;IL-4, interleukin-4; MEN, menadione; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; OD, optical density; PMS, phenazine methosulfate; SD, standard deviation; SDS, sodium dodecyl sulfate; XTT, sodium 3'-[1-[(phenylamino)-carbonyl]3,4-tetrazolium]-b/s (4-methoxy-6-nitro)benzene-sulfonicacid hydrate.
zolium salt MTI" have proven to be extremely useful (Mosmann, 1983; Cole, 1986; Gerlier and Thomasset, 1986; Tada et al., 1986; Ferrari et al., 1990). Advantages of the M T T colorimetric assay over more conventional techniques such as incorporation of [3H]thymidine, include savings in the cost of reagents and equipment, reduced labor by elimination of sample processing steps required for liquid scintillation counting, as well as avoiding problems of safety and waste disposal associated with the use of radioisotopes. One weakness inherent in the use of M T T is that the resulting colored formazan product is insoluble, precluding direct spectrophotometric
258 absorbance measurements without first dissolving the crystals. While a number of useful procedures have been devised to handle this problem, they all require additional sample processing steps and increase the length of time required to complete the assay (Mosmann, 1983; Tada et al., 1986; Hansen et al., 1989; Niks and Otto, 1990). A new tetrazolium salt, XTT, has been synthesized by Paull and colleagues (1988). Bioreduction of XTT yields a highly colored formazan product, which in contrast to other tetrazolium salts like Mq"F, is water soluble. Studies by Scudiero et al. (1988) evaluated the use of XTI ~with human tumor cells and demonstrated the ability of electron coupling agents to potentiate bioreduction of XTT. In this paper we examined the use of XTT in combination with two such agents, menadione and phenazine methosulfate, in a number of common cytokine dependent proliferation assays. Side by side comparisons with MTT indicated that under optimal conditions XTT offers a high degree of sensitivity, while offering considerable savings in time and labor by eliminating the need to solubilize the formazan product prior to absorbance measurements.
Materials and methods
Reagents XT-F was purchased from Diagnostics Chemicals, Charlottetown, P.E.I., Canada. Phenazine methosulfate was purchased from Aldrich Chemical Company, Milwaukee, WI. MTT and menadione were purchased from Sigma Chemical Company, St. Louis, MO. Concanavalin A (ConA) was purchased from ICN ImmunoBiologicals, Lisle, IL. Recombinant human interleukin-2 (IL2) and recombinant murine interleukin-4 (IL-4) were produced and purified at Lilly Research Laboratories. Murine cell lines HT-2, an IL-2 dependent T cell line and FS84.1, a T cell hybridoma which produces IL-2, were kindly provided by John Kappler and Philippa Marrack, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO. HT-2 was maintained by serial passage in
medium supplemented with a ConA induced culture supernatant from FS8-4.1. M C / 9 an IL-3 dependent mast cell line, was obtained from the American Type Culture Collection, Rockville, MD. NFS-60, an IL-3 dependent myeloid leukemia, was kindly provided by James Ihle, St. Jude Children's Research Hospital, Memphis, TN. 11.6 is an IL-4 dependent T cell line derived by A.G. as a variant of an antigen specific T cell clone. D10.G4 is an antigen specific T cell clone, kindly provided by Charles Janeway, Yale University School of Medicine, New Haven, CT. MC/9, NFS-60 and 11.6 were maintained by serial passage in media supplemented with a ConA induced culture supernatant from D10.G4. Mice (C57BL/6 X DBA/2)F 1 mice used in these studies were purchased from Charles River Laboratories (Portage, MI). Animal care and use was in accordance with guidelines of Lilly Research Laboratories and the American Association for Laboratory Animal Care. Colorimetric assays The medium used for all experiments was Dulbecco's modified Eagle medium supplemented with 20 /~g/ml gentamicin sulfate, 100 U / m l penicillin G, 100 ~ g / m l streptomycin sulfate, 2 mM L-glutamine (Gibco Laboratories, Grand Island, NY), 10% fetal bovine serum (HyClone Laboratories, Logan, UT), and 0.05 mM 2mercaptoethanol (Sigma). Unless specifically indicated, cells were cultured in a volume of 100 ~1, in flat bottom 96 well tissue culture plates (Costar, Cambridge, MA) at 37°C in 10% C O 2 and air. Mitogen stimulated spleen cells were cultured at 4 x 105 per well in 4/.tg/ml ConA. Factor dependent cell lines were cultured at 4 × 103 cells per well. After 40 h of incubation with the cell lines or 64 h with ConA activated splenic T cells, the cultures were pulsed for 7-8 h with either XTT (alone or in combination with PMS or MEN) or MT-F. The procedure used for the MTF assay was based on that previously described by Tada and colleagues (1986). MTT solutions were prepared at 5 mg/ml in phosphate buffered saline, filter sterilized and stored in the dark at 4 ° C. Mq-'-F
259 solutions were discarded after 1 month. 2 0 / z l of MTT at 5 m g / m l was added to each 100 p,l culture. After incubation for an additional 7 - 8 h at 37 ° C the formazan crystals were dissolved by addition of 100 /zl 10% sodium dodecyl sulfate (SDS) in 0.01 N HCI. The plates were then incubated over night and the optical density (OD) of the wells determined using a Molecular Devices Corp. UVmax kinetic microplate reader at a test wavelength of 570 nm and a reference wavelength of 690 nm. Each plate contained 'blank' background control wells containing an appropriate volume of media, MTI" and acidified SDS, but no ceils. x T r solutions were made fresh each day by dissolving XTI" in hot media at 1 m g / m l . This was most easily accomplished by heating a tube containing medium in a 60 ° C waterbath. When the medium was heated sufficiently added X T T would completely dissolve. PMS was made up as 100 mM solution in phosphate buffered saline and stored at 4 ° C for periods up to 1 month. M E N was made up fresh each day as a 100 mM solution in dimethyl sulfoxide. PMS and M E N commonly required sonication to completely dissolve. PMS or M E N were added to the x T r solution immediately before use. 25/zl of X T T / P M S or X T T / M E N were added to each 100/zl culture. Unless specifically indicated, PMS was used at a final concentration of 25/xM. After an additional 7 - 8 h of incubation at 3 7 ° C the
O D of the wells was determined using a UVm~x kinetic microplate reader at a test wavelength of 450 nm and a reference wavelength of 650 nm. Each plate contained appropriate blank control wells containing media, X T T and PMS or MEN, but no cells.
Results
Factor dependent cell lines have proven to be valuable research tools as indicators of cell activation leading to cytokine production as well as in the quantitation of specific cytokines (Gillis et al., 1978; Kappler et al., 1981; Yokota et al., 1984; Lee et al., 1986; Tada et al., 1986; Weinstein et al., 1986). The IL-2 dependent T cell line HT-2 has proven to be particularly useful in this regard and we began our examination of X T T with these cells. Previous studies had demonstrated that many human tumor lines do not metabolize X T T efficiently unless electron coupling agents are added (Scudiero et al., 1988). In our initial studies we evaluated the ability of PMS and M E N to enhance bioreduction of X T T by HT-2 cells proliferating in response tO recombinant IL-2 or IL-4. Table I summarizes the data from several similar experiments in which HT-2 cells proliferating in response to optimal concentrations of IL-2, were pulsed with X T T plus increasing concentra-
TABLE I ANALYSIS OPTIMAL MEN AND PMS CONCENTRATIONSFOR XTT LABEL OF HT-2 CELLS PROLIFERATING IN RESPONSE TO IL-2 HT-2
Blank
HT-2 alone
HT-2 clL-2
Difference exp-cntrl
XTT alone PMS/~M 200 100 50 25 12.5 6.25 3.12 1.56 0.78
0.173
0.172
0.308
0.136
0.839 0.584 0.440 0.344 0.278 0.236 0.212 0.193 0.182
0.959 0.684 0.583 0.459 0.338 0.281 0.238 0.203 0.185
1.940 1.986 1.989 2.045 1.885 1.844 1.497 0.921 0.486
0.981 1.302 1.406 1.586 1.547 1.563 1.259 0.718 0.301
XTT alone MEN/zM 200 100 50 25 12.5 6.25 3.12 1.56 0.78
Blank
HT-2 alone
HT-2 clL-2
Difference exp-cntrl
0.169
0.170
0.296
0.126
0.380 0.297 0.248 0.211 0.195 0.187 0.177 0.174 0.169
0.402 0.318 0.265 0.230 0.210 0.198 0.185 0.179 0.175
0.923 0.787 0.784 0.828 0.723 0.538 0.415 0.358 0.326
0.521 0.469 0.519 0.598 0.513 0.340 0.230 0.179 0.151
260 4.0
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Fig. 1. Absorbance spectra of X T T / P M S substrate mixture in culture media at time zero ( . . . . . . ) as compared to that of the X T T formazan product released into the supernatant by proliferating HT-2 cells cultured with X T T / P M S ( ).
tions of PMS or MEN. Examination of the absorbance values in the background blank controls revealed that the addition of PMS or M E N to X T T directly leads to a concentration dependent increase in absorbance values. However at low concentrations the inclusion of either PMS or M E N led to an even greater potentiation of X T T bioreduction by proliferating HT-2 cells. Comparing the difference in absorbance values generated by HT-2 cells in the presence or absence of IL-2, the largest differential occurred at PMS or M E N concentrations of 25 ~ M . At these concentrations background blank absorbance values were reasonably low. Responses generated with Xq"-F used in combination with PMS were substantially greater than those with MEN, albeit with a higher background. Similar results were obtained with HT-2 cells proliferating in response to IL-4 (data not shown). Fig. 1 depicts absorption spectra of the X T T / P M S substrate in culture medium as compared to that of the bioreduced formazan product released into the supernatant by HT-2 cells. The X T T formazan product had an absorbance maxim u m at wavelengths between 440-490 nm. In contrast, the medium X q - T / P M S substrate mixture had a low absorbance in this wavelength
range. Accordingly a test wavelength of 450 nm was selected. This choice eliminated the need to acidify the cultures because of phenol red interference as occurs with M T T at 570 rim. A reference wavelength of 650 nm was chosen where absorbance of substrate and product were negligible. Having determined an optimal concentration of PMS to use with X T T in measuring proliferation by HT-2 cells, we made side by side comparisons with MTI" and examined the kinetics of bioreduction with the two tetrazolium salts. In a representative experiment depicted in Fig. 2, recombinant human IL-2 or recombinant murine IL-4 were titrated into replicate cultures of HT-2 cells. After 40 h of incubation the wells were pulsed with either M T T or X T T plus PMS (25 ~ M ) and incubated an additional 8 h. The M T T formazan crystals were dissolved with acidified SDS and the O D measured at 570 nm, while the XTI" pulsed plates were read directly at 450 nm without further manipulation. The background blank absorbance values observed with X T I ' / PMS were greater than those observed with MTF. However even after subtracting the blank absorbance values, the signal generated using X T T / P M S was reproducibly greater than that observed with MTT. Conversely the signal-tonoise ratio in the proliferative response to IL-2 was much greater using MTT, while the ratios with XT-F and M T T were comparable when the stimulus was IL-4. When the specific activity of the cytokine preparations was determined based on linear regression analysis of the linear portions of the titration curves and defining a unit of activity as the square root of the maximum multiplied by the minimum response, the specific activity of both the IL-2 and IL-4 preparations observed with X T T / P M S readouts were reproducibly greater than or equal to those observed with MTT. Fig. 3 depicts the kinetics of bioreduction of X T T / P M S and M T T by proliferating HT-2 cells. With M T T the reaction was extremely rapid. The formation of the f o r m a z a n product was at 90% of maximum within 2 h and reached its plateau in 3 h. In contrast proliferating HT-2 cells continued to metabolize X T T (used with PMS) for periods longer than 8 h. Thus it appears that while the
261 2.0
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values c o m p a r a b l e to those o b s e r v e d with M T T required at least a 4 h pulse and longer labeling times w e r e necessary to give greater values. W e w i s h e d to d e t e r m i n e w h e t h e r the results w e had o b s e r v e d with H T - 2 w e r e generally applicable to other factor d e p e n d e n t cell lines. For this p u r p o s e w e e x a m i n e d the r e s p o n s e of a seco n d T cell line 11.6 which proliferates in r e s p o n s e to IL-4, but not IL-2, the mast cell line M C / 9 which proliferates in r e s p o n s e to IL-3 and IL-4 ( L e e et al., 1986) and the m y e l o i d l e u k e m i a N F S 60 which proliferates in r e s p o n s e to IL-3 and
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rate of f o r m a z a n g e n e r a t i o n with M T F is faster, the cells are able to m e t a b o l i z e X T T for longer periods of time. This difference m a y account for the higher absorbance values observed with X T T / P M S , as d e t e r m i n e d by c o m p a r i s o n s o f the absolute difference b e t w e e n positive and negative controls. T o achieve X T £ f o r m a z a n absorbance
i
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Fig. 2. Comparison of XTT/PMS (•) and MTT (o) colorimetric measurements of the proliferative response of HT-2 cells cultured with increasing concentrations of (A) recombinant human IL-2 or (B) recombinant murine IL-4. The mean-l-SD of the background blank controls were: (A) XTI'/PMS 0.414 +0.013 and MTr 0.259+ 0.005; (B) XTT/PMS 0.420+ 0.006 and MTT 0.255 +0.006. The background blank values were subtracted from the data.
i
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HT-2 + IL-4 H T - 2 ALONE BLANK
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MTT LABELING TIME IN H O U R S
Fig. 3. Time course of bioreduction of XTT/PMS and MTT by proliferating HT-2 cells. Replicate cultures containing media alone (blank), HT-2 cells alone or HT-2 plus IL-4 were pulsed with (A) XTT/PMS or (B) MTT and then harvested at the indicated time points. Note: culture volume of XTT assay was increased to 200/~l to reduce potential problems of cell over growth.
262
GM-CSF (Weinstein et al., 1986). Proliferation of both M C / 9 and NFS,60 were induced using ConA stimulated culture supernatants from the helper T cell clone D10.G4, which contains IL-3, IL-4 and GM-CSF among other cytokines. The T cell line 11.6 was cultured with recombinant
murine IL-4. In each case the cells were cultured alone or in the presence of saturating cytokine preparations and then tested for x T r reduction in the presence of increasing concentrations of PMS or MEN. The results from several similar experiments
T A B L E II A N A L Y S I S O P T I M A L PMS A N D M E N C O N C E N T R A T I O N S F O R X T T L A B E L (A) NFS-60 proliferating in response to D10.G4 C o n A supernatant Blank X T T alone PMS/xM 200 100 50 25 12.5 6.25 3.12 1.56 0.78
NFS-60 alone
NFS-60 D10 CAS
Difference exp-cntrl
0.173
0.178
0.349
0.171
0.839 0.584 0.440 0.344 0.278 0.236 0.212 0.193 0.182
0.931 0.728 0.580 0.557 0.402 0.289 0.237 0.210 0.191
1.965 2.012 1.992 1.987 2.010 1.935 1.644 0.835 0.464
1.034 1.284 1.412 1.430 1.608 1.646 1.407 0.625 0.273
Blank X T T alone MEN/xM 200 100 50 25 12.5 6.25 3.12 1.56 0.78
NFS-60 alone
NFS-60 D10 CAS
exp-cntrl
0.169
0.171
0.345
0.174
0.380 0.297 0.248 0.211 0.195 0.187 0.177 0.174 0.169
0.388 0.330 0.298 0.294 0.328 0.314 0.296 0.272 0.230
1.682 1.783 1.922 1.958 1.903 1.680 1.280 0.874 0.581
1.294 1.453 1.624 1.664 1.575 1.366 0.984 0.602 0.351
Blank
MC/9 alone
MC/9 D10 CAS
exp-cntrl
0.169
0.159
0.378
0.219
0.380 0.297 0.248 0.211 0.195 0.187 0.177 0.174 0.169
0.374 0.289 0.239 0.206 0.188 0.178 0.170 0.162 0.161
1.705 1.802 1.882 1.661 1.406 1.085 0.753 0.563 0.471
1.331 1.513 1.643 1.455 1.218 0.907 0.583 0.401 0.310
Blank
11.6 alone
11.6 clL-4
Difference
0.169
0.166
0.324
0.158
0.380 0.297 0.248 0.211 0.195 0.187 0.177 0.174 0.169
0.365 0.282 0.239 0.209 0.191 0.182 0.176 0.172 0.165
1.552 1.559 1.811 1.895 1.861 1.624 1.306 0.960 0.669
1.187 1.277 1.572 1.686 1.670 1.442 1.130 0.788 0.504
Difference
(B) M C / 9 proliferating in response to D10.G4 C o n A supernatant Blank X T T alone PMS g M 200 100 50 25 12.5 6.25 3.12 1.56 0.78
MC/9 alone
MC/9 D10 CAS
Difference exp-cntrl
0.173
0.168
0.400
0.232
0.839 0.584 0.440 0.344 0.278 0.236 0.212 0.193 0.182
0.818 0.587 0.443 0.339 0.272 0.235 0.210 0.187 0.178
2.057 2.049 2.039 1.946 1.819 1.635 0.986 0.571 0.455
1.470 1.606 1.700 1.674 1.584 1.425 0.799 0.393 0.277
X T T alone MEN/zM 200 100 50 25 12.5 6.25 3.12 1.56 0.78
Difference
(C) 11.6 Proliferating in response to IL-4 Blank X T T alone PMS/zM 200 100
50 25 12.5 6.25 3.12 1.56 0.78
11.6 alone
11.6 clL-4
Difference exp-cntrl
0.173
0.172
0.319
0.147
0.839 0.584 0.440 0.344 0.278 0.236 0.212 0.193 0.182
0.809 0.573 0.435 0.348 0.279 0.235 0.208 0.184 0.179
1.773 1.820 1.902 1.926 1.613 1.347 1.106 0.644 0.414
0.964 1.329 1.491 1.265 1.068 0.871 0.436 0.230 0.235
X T T alone M E N ~tM 200 100 50 25 12.5 6.25 3.12 1.56 0.78
exp-cntrl
263
are summarized in Table II. In contrast to our observations with HT-2, MEN was very effective at potentiating metabolism of XTT with each of these cell lines, proving to be equal to PMS. To determine the optimal working concentrations of PMS and MEN we again compared the absolute
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Fig. 5. Comparison XTT/PMS and MTT colorimetric measurements of proliferation by graded numbers of ConA activated splenic T cells. The mean +SD of the blank control values for XTT/PMS were 0.425 + 0.006 and for M'IT 0.288 +0.012. The background blank values were subtracted from the data.
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CYTOKINE
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Fig. 4. Comparison of XTT/PMS (e) and MTT ( o ) colorimetric measurements of lymphokine dependent proliferation by: (A) the myeloid leukemia NFS-60 cultured with increasing concentrations of D10.G4 ConA supernatant (D10 CAS), (B) the mast cell line MC/9 cultured with increasing concentrations of D10 CAS and (C) the T cell line 11.6 cultured with increasing concentrations of recombinant IL-4. The mean + SD of the blank control values were: (A) XTT/PMS 0.450 :t: 0.015 and MTT 0.226-t-0.009; (B) XTT/PMS 0.443+0.010 and MTT 0.239 + 0.011 and (C) XTT/PMS 0.448 + 0.010 and MTT 0.234 :t: 0.009. The background blank values were subtracted from the data.
difference in formazan OD values in the presence and absence of saturating concentrations of the cytokines. Using this method with NFS-60 the optimum concentrations were 6.25/zM PMS and 25/~M MEN, the results being very similar. With M C / 9 50 /~M was optimal for both PMS and MEN, the background values being slightly higher with PMS. With the T cell line 11.6 50/zM PMS and 25 /xM MEN were optimal and the results with MEN were superior. Having established that each of these cell lines was capable of reducing XTT in the presence of PMS, we made side by side comparisons with MTT. Replicate plates were set up in which appropriate cytokine preparations were titrated into cultures of the factor dependent cell lines. Following 40 h of incubation the plates were pulsed with either Mq~F or XTT plus PMS (25/xM). As shown in a representative experiment depicted in Fig. 4, each of these cell lines generated higher formazan absorbance values with XTT/PMS as compared to those with MTT. The signal-to-noise ratios were slightly greater using MTT with NFS60 and M C / 9 and slightly greater using XTT with 11.6. The specific activities of each of the cytokine preparations observed with these cell
264 TABLE III ANALYSIS OPTIMAL PMS AND MEN CONCENTRATIONS FOR XTT LABEL OF ConA STIMULATED SPLEEN CELLS
XTT alone PMS ~ M 200 100 50 25 12.5 6.25 3.12 1.56 0.78
Blank
Spleen alone
Spleen ConA
Difference exp-cntrl
0.197
0.244
0.372
0.128
0.941 0.681 0.511 0.374 0.327 0.278 0.246 0.226 0.211
1.187 0.965 0.925 0.886 0.896 0.760 0.617 0.438 0.317
1.933 1.859 1.925 1.762 1.555 1.447 1.395 1.057 0.624
0.746 0.894 1.000 0.876 0.659 0.687 0.778 0.619 0.307
lines using X q - T / P M S were reproducibly greater than or equal to those observed with MTT. The last system examined was the proliferative response of normal splenic T cells stimulated by ConA. The ability of PMS and MEN to potentiate bioreduction of X T T was evaluated by adding increasing concentrations of these agents plus X T T to replicate cultures of splenic T cells which had been incubated in the presence or absence of ConA. Results from several similar experiments are summarized in Table III. The maximum X T T formazan O D difference between ConA stimulated cultures and controls was observed with PMS at 50 /xM. The absorbance values with X T T / M E N pulsed cells were substantially lower. Using this information the X T T / P M S and M T T colorimetric assays were compared by measuring the formazan absorbance values generated by graded numbers of ConA induced splenic T cell blasts. A representative experiment is shown in Fig. 5. With both tetrazolium salts there was a good correlation between cell number and the resulting formazan absorbance values. While the dynamic range was slightly greater with M T T than with X T T / P M S , at cell concentrations below 5 x.106/ml more formazan product was formed per cell with X T T than with MTT.
Discussion
These studies demonstrate that results with the tetrazolium salt X T T compare favorably to
XTT alone MEN izM 200 100 50 25 12.5 6.25 3.12 1.56 0.78
Blank
Spleen alone
Spleen ConA
Difference exp-cntrl
0.187
0.233
0.362
0.129
0.420 0.332 0.279 0.242 0.223 0.205 0.197 0.193 0.188
0.545 0.473 0.435 0.400 0.391 0.380 0.355 0.328 0.307
1.207 1.088 1.103 1.000 0.767 0.609 0.505 0.437 0.403
0.662 0.615 0.668 0.600 0.376 0.229 0.150 0.109 0.096
those with M T T when used for colorimetric measurements of lymphokine dependent cell proliferation, while offering significant advantages. X T T can be reduced by dehydrogenase enzymes of viable ceils yielding a highly colored, water soluble formazan product. Thus in contrast to other tetrazolium salts, X T T offers the advantage of eliminating the need for formazan crystal solubilization prior to multiwell scanning spectrophotometric measurements, thereby reducing both labor and assay time. One added complication is that most cells do not metabolize X T T efficiently unless an electron coupling agent is added. This was true for all the murine cells we examined. To varying degrees PMS and MEN each demonstrated the ability to enhance bioreduction of X T T by different lymphoid and myeloid cells. With the mast cell line M C / 9 , the myeloid leukemia NFS-60 and the T cell line 11.6, MEN and PMS were almost equally effective in potentiating bioreduction of XTT. In contrast with normal T cell blasts and the T cell line HT-2, PMS was significantly more effective than MEN. At first glance it may seen disheartening that the optimal response of each of these ceils occurred at different concentrations of MEN or PMS. However, it is noteworthy that entirely satisfactory results were obtained with each of these cell lines as well as mitogen activated T cells using PMS at 25 ~M. In fact all the side by side comparisons of X T T and MTI" described in this paper were made using PMS at this concentra-
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tion. We have also observed good formazan production using XT-F in combination with PMS by mouse peritoneal macrophages, the m a c r o p h a g e tumor line P388D t, normal murine B lymphocytes stimulated with IL-4 plus anti-IgM antibody and with the human histiocytic lymphoma U-937 (data not shown). The final procedure which we found to be most generally applicable was as follows. On the day of assay X T T was dissolved in hot media at a concentration of 1 m g / m l . Immediately before use, PMS (100 m M stock in saline stored at 4 ° C) was added to the X T F solution giving a PMS concentration of 125/zM. 25/xl of this X T T / P M S solution was added per 100 /xl culture giving a final concentration of 0.2 m g / m l XT]? and 25 /zM PMS. After incubation at 37 o C for 4 - 8 h the O D of the cultures was determined using a test wavelength of 450 nm and a reference wavelength of 650 nm, subtracting blank control values. W h e n applied to specific cell lines, lower background absorbance values a n d / o r higher signal to noise ratios may be obtained using M E N or PMS at other concentrations. An additional feature of X T T may be of potential use in certain circumstances. Based on the kinetics of formazan production, cells a p p e a r to remain metabolically active for longer periods when pulsed with X T T / P M S than with MT-F. This feature may be of value in cytotoxicity assays or in studies of cell activation. This fact may also contribute to the higher formazan absorbance values observed with X T - F / P M S as c o m p a r e d to MTI'.
Acknowledgement We would like to express our appreciation to Noel Richard for bringing the use of X T T to our attention.
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