A simple, rapid and sensitive fluorimetric assay for the measurement of cell-mediated cytotoxicity

EISWIER

Journal of ImmunologicalMethcdr I85 (1995) W-208

A simple, rapid and sensitive fluorimetric assay for the measurement of cell-mediated cytotoxicity

A fluorimetric method using Cmethylumbelliferyl heptanoate (MfJH) has been developed for detecting cellmediated cytotoxicity and cell proliferation. The assay is bared on the hydrolysis of the fluorochrome (MUH) by intracellular e~terasesof viable cells resulting in the production of highly fluorescent 4.methylumbelliferone that can be measured in a micro~late fluorimeter. Because of a similarity to the princ$k of the widely used calorimetric MTI assay, a ccmtpari& was made between the two assays when me~s&g cell prolifer&n and LAK cell cytotoxicity to different target cell types. The results have shown that the MUH assay represents a method for evaluating both cell-mediated cytotoxicity and cell proliferation which is campletely comparable to the M’IT method. The rapidity of the new wtotaxicity assay, 5 h in contrast to 9 h for the MTI’ assay, its applicability to both adherently and nonadherently graving target cells and its high accuracy due to the avoidance of centrifugation steps make this method a serious antender for replacing conventional radioactive techniques. Keywords; 4-Metbylumbelliferyl killer cell; Tumor cell line

heptanaate; Fluorimetric assay; WI-mediated

cytotoxicity; Lymphokine-activated

basic immunological functions. Radioactive chromium (“Cr) release (Brunner et al., 1%8) is Methods tensively

utilizing

radioactive

used for the

isotopes are ex-

measurement

of many

Abbreviationr AFU, absotutefluorescenceunit% CMC, c&mediated cytotoxtcity:CT, crtotoxicily;CV, meffcisnt of varialiaa\; OMSO. dimethylsulfoxide:EL4SN. supernalantof PMA stimulated EL4 c&s; E:T ratio, effector/ta,get cell ratio; KS, fetal calf sawn: L4K cells. lymphok%-activated killer cells; MUH. 4-me&hylumbelliferylhcptanoate:MlT, U-l4~-dimethylthiazat-2-yt~~5-di~h~~ytlel~~li~~ bromide; PMA, phorbot t2-“wristate t3acetate; PBS. phosphatebuffered raline.

’ TelJFax: <3652)-417-159.

for example

still regarded

as the gold standard

for determining cell-mediated cytototicity (CM0 The use of the chromium release method, however, like any other radioactive technique, has several disadvantages. In addition to the potential danger of chronic low dose radiation and problems of handling and disposal, the low sensitivity and the high spontaneous chromium release that often occur have prompted researchers to look for new methods to measure cell-mediated cytotoxicity. Bearing all these major drawbacks in mind, a

LW2Z1759/95/509.500 1995Elrevier ScienceB.V. All rightsreselvcd ssD,0022-1759~95~00t15-8

of calorimetric and fluorimetric assays have been developed to replace radioactive techniques. Assays based on dye exclusion (Larsson and Nygren, 1989) or fluoroehrome release (Kolber et al., 198& Blomberg and Ulfstedt, 1993; Lichtenfels et al., 1994) and assays measuring inhibition of metabolic activities &arssou and Nygren, 1989; Kriiger-Krasagakes et al., 1992) have been reported. Detecting different stages in the death of target cells - e.g., decrease of metabolic activity, increased membrane permeability or detachment of adherent cells from the surface -provides methods which differ iu efftcacy and often yield considerably different cyt+ toxicity values. These differences could be further increased using different detection techniques (e.g., radioisotopic, calorimetric, fluorimetric or tlow cytometric). Most of these assays have either proven to be time-consuming or required expensive equipment such as a flow cytometer or a time resolved fluorimeter. Since fluorimetric methods excel in sensitivity and metabolic activity is regarded as the most fundamental indicator of viability at the cellular level, a fluorimetric assay based on the measurement of metabolic function may be suitable for estimating CMC. Dotsika and Sanderson (1987) have tested six 4-methylumbelliietyl esters for their ability to determine cell viability. These fluorogen substrates readily diffuse through the cell membrane and are hydrolysed by intracellular esterases or sulphatases resulting in the pmduction of highly fluorescent 4-methvlumbelliferone. 4methvlumbelliieryl heptanoate (MUHI has proven to be superior among the six candidates with respect to providing sufficient activity and a low background. MUH assa)s for determining proliferation of keratinocytes (Stadler et al., 1989) and human dermal microvascular endothelial cells (Hettmannsperger et al., 1993; Detmar et al., 1990). as well as for testina the effect of antioroliiera&e compounds on human melanoma cells (Zouboulis et al., 1991) have subsequently been published. However only one attempt has been made to adapt this method for the measurement of cell-mediated cytotoxicity (Kritger-Krasagakes et al., 1992). In this publication the determination of LAK cell cytotoxicity against adherent target number

cell lines was reported. The authors emphasized, however, that the method was able to measure CMC to adherent targets only, beeause the eriterion of cell death in their assay was the detachment of adherent target cells from the surface. The production of a fluorescent compound from a fluorogen substrate in the above assay is rather similar to the widely used calorimetric MTI assav (Mosmann. 1983). where a tetrazolium salt is cleaved and converted to a blue formazan by the mitochondrial dehydrogenases of living cells. The MTT assay can be applied to both adherently and non-adherently growing cells. Recognizing the similarity in principle between the calorimetric MTI’ assay and the fluorimetric MUH assay (both methods are based on measurement of metabolic dctivities of surviving target cells), we have developed a fast and sensitive fluorescent assay, capable of measuring CMC against both adherent and non-adherent target cells. This new method combines the well proven MTT assay with the high sensitivity of fluorescent techniques.

2.

Materials and metbuds

Cmethylumbelliietyl heptanoate (MUH) was purchased from Sigma. A stock solution of MUH (10 mg/ml in DMSO) was diluted in phosphatebuffered saline (PBS, pH 7.3) to give a concentration of 3W pg/ml (3 X MLJH) for cytotoxicity assays or 100 pg/ml (MUH working solution) for proliferation assays. M’IT (3_[4,Sdimethylthiazol2-yl]-2,5-diphenyltetrazolium bromideXSigma) was dissolved in PBS at 5 mg/ml WIT stock solution) and sterilized by filtering. RPM1 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Sebak, Aidenbach, Germany), 10 mM glutamine and 40 pg/ml gentamicin (Chinoin, Budapest, Hungaty) was used as a culture medium for all cell lines, (subsequently designated as complete medium,). Round bottom 96 well microtiter plates (Nunc, Roskilde, Denmark) were used for all the assays.

2.2. AnimaLc

Cells were removed by centrifugation (800 x g, 15 min) and aliquots stored at - 70°C until use.

AKR/J and BALB/c mice were bred and maintained in the animal house of the Department of Pathophysiology of University Medical School Debrecen, and were allowed free access to food and water. Female mice were used at the age of 8-14 weeks. 23. Cell lines The following adherent, weakly adherent and non-adherently growing cell lines served as target cells in the cytotoxicity assays. Adherent cell lines, B16/Fl and B16/FlO, are melanoma cells of C5781/6 origin. The Hep-2 line originated from a human epithelioma. Weakly adherent cell lines, Ta3Ha (Hauschka et al., 1971) and 410.4 (Blazar et al., 1980). were derived from spontaneous mouse mammary adenocarcinomas of A/HeHa and BALB/cfC3H ori&, respectively. Non-adherent cell lines, Ta3St (non-adherent subline of Ta3 tumors) and Bw5147 thymoma line (AKR/J), were also used. The CIXL (IGZdependent T cell line) was used for proliferation assays and ELA thymoma cells for the generation of ILZcontaining conditioned medium. All the cell lines were grown in complete RPM1 1640 medium in a 5% CO, atmosphere. 2.4. Production

of EL4

cell supernatont (EL4SN)

Stimulation of EL4 cells in order to gain IL2containing conditioned medium was carried out as previously described (Farrar et al., 1980) with slight modifications. Briefly, EL4 thymoma cells in the logarithmic growing phase were collected and cultured for 4 h in complete medium cantaining 20 ng/ml PMA in a humidified 37°C. 5% CO2 incubator. The cells were then collected and washed 3 times with RPM1 1640 medium. Washing the cells effectively removed residual PMA. Following the washing steps, cells were resuspended in complete medium and dispensed in 24 well cell culture plates (Nunc, Roskilde, Denmark) at a densitv of 10’ cells/ml. After incubation ior 48 h in a- humidified 37°C. 5% CO, incubator, culture supernatant was collected.

LAK cells were generated as described (Groscourth et al., 1990). Briefly, mononuclear cells from the spleens of AKR/J mice (for most of the Cl’ cultures) or from BALB/c mice (for the Bw5147 CT assay) were separated on a Ficoll-Ummiro (1.080 g/ml) gradient. The iuterface cells were collected and washed three times with RPM1 1640 medium. Adherent cells were removed by allowing them to adhere to petri dishes during 1 h of incubation, in a humidified 37”C, 5% CO, incubator. Non-adherent cells were then cultured for 72 h in complete medium containing 20% conditioned medium of PMAactivated EL4 cells (EIASN). Cells were washed twice with PBS (pH 7.3) before use in the cytotoxicity assays.

In order to avoid high background fluotescence caused by the phenol red content of the culture medium, effector and target cells were resuspended in modified coIourless RPM1 1640 medium without phenol red supplemented with 10 mM glutamine and 40 pg/ml gentamicin. 2 X IO’ target cells/well were seeded in 96 well round bottom microplates in a volume of 50 pl. Effector cells were then added in 50 pl to give effectortarget cell ratios of 5:l. l&l, 20~1 and 50~1 (for 4 h assays) or 1:2, l:l, 2~1, 5:l and lo:1 (for 16 h assays), respectively. At each ET ratio eight replicates of the co-culture as well as eight replicates of the effector cells alone were set up in parallel. In each experiment eight replicates of target cells alone were also set up. The plates were kept at 37°C in a humidified 5% CO, incubator for 4 h (or in some experiments for 16 h, as indicated). 50 pl of 3 x MUH solution were then added to each well. Following 30 min incubation at 37”C, fluorescence in each well was measured usina a Fluoroskan II Neonat microolate fluorimeter (Labsystems, Helsinki, Finland) equipped with a xenon lamp and fitted with 355 nm excita-

tion and 460 om emission filters. Fh~oroskan II has been shown to detect 0.1 pmol of 4-methylumbelliferone (data of the manufacturer). Since the MUH is added to the target-effector cell mixture, absolute fluorescence units (AFU) must be corrected for the fluorescence caused by the effector cells (Table 1). Therefore the percentage specific killing was calculated using the following formula: %killing = 1oLlx (AFU of target cells alone -(test

AFU - AFU of effector cel!s alone)]

x {AFU of target cells alone} - ’ where (test AFU - AFLI of et?ector cells alone) represents the fluorescence of the surviving targets and consequently the numerator mirrors the number of the killed target cells. For a better illustration of the calculation an example is given presenting original values obtained at the highest ET ratio using Ta3Ha cells as target: AFU,,, = 851, AFU,,,.,

= 1534, AF&_,,

were incubated together in eight replicates for 4 h in lo(i pl modified colourless RPM1 1640 (37”C, 5% CO,, humidified incubator) using the same conditions as described for the MUH assay. 10 ~1 of stock m solution were then added to each well followed by another 4 h incubation (37”C, 5% Co,) for Mm reduction. At the termination of the assay, cells were pelleted by centrifogation and after discarding the supematant, 100 ~1 of acidified isopropanol (containing 0.004 M HCI) were added to all wells and mixed thoroughly to dissolve the dark bloc crystals. After 30 min at room temperature. plates were read by a Multiskan Multisoft microplate photometer (Labsys terns, Helsinki, Finland) using a test wavelength of 540 nm and a reference wavelength of 690 nm. Percentage cytotosicity values were calculated as follows: %killing = 100 x (OD of target cells alone -(test

OD - OD of effector cells alone))

x [OD of target cells alone] -’

= 729

where OD = optical density. All results were expressed as mean values and standard deviations of at least quadruplicate experimmts.

1534 - (&Xl- 729) %killing = 100 x ______ = 92.05% 1534

2.8. CTLL proliferation may This assay was performed as previously described (Hussain et al.. 1993) with some minor modifications. Briefly. effector and target cells

IL-2-dependent CXLL cells (104/well) were added in four replicates to round bottom 96 well

Absolute fluorescence “nits TdHa 151.16*9.97 234.79 f 10.98 297.38 f 17.25 678.56 f 25.64

Bvm47 96.18f 1.48 109.74 k 4.46 fSS.08 f 6.63 282.43 + 14.94

410.4 132.14 * 2.87 165.48 + 3.56 243.26 !c 7.50 394.01 + 15.94

HepZ 150.59 * 20731-t 3,553* 536.1Of

4.89 690 12.w 16.89

microtiter plates which had previously recieved 200 pl complete medium containing 0. 3. 6.25, 12.5,25 and 50% of EL&N. After 48 h of incubation, cultures were measured in both assays as follows. 1. After the addition of 20 (I stock MlT solution to all wells of the plate, the MTT assay was terminated as described. 2. For the MUH assay, plates were centrifuged (450 xg, 5 min) and washed once with 200 pl PBS to remove FCS-containing medium. 100 ~1 MUH working solution were then added to each well followed by 30 min of incubation and measurement as described. 2.9. FCS proliferationassay Quadruplicates of a double serial dilution of FCS were prepared with RPM1 1640 medium using volumes of 100 &l/well in 96 well round bottom microtiter plates. 410.4 adenocarcinoma cells were then seeded in 100 ~1 of the same medium in each well at 2 density of 5 X 103/well. The final concentrations of FCS obtained as a result of this procedure were 50, 25, 12.5, 6.25, 3 ana 0%. After an incubation of 48 h in a humidified 37”C, 5% Co, incubator, plates were tentrifuged (450 X g, 5 min), washed twice with PBS

and assay cultures were terminated in both assays as described.

3. Ftesults

Since a high degree of correlation between cell number and absolute fluorescence values is essential for fluorimetric assays measuring viable cell numbers, we have investigated the relationship between these parameters. A serial hvo-fold dilution using four different cell lines (Ta3Ha, Bw5147, 410.4 and Hep-2) was made in colorless medium and MUH activity was determined after 4 h of incubation at 31°C. A direct correlation was found between these two variables over a wide ranee of cell numbers with r values of 0.985 (p
testing and cloning could prevent segregation of specific cells.

Since lymphokine-activated ki:ler &AK) cells are easy to generate and are known to exert a strong in vitro cytotoxic effect cm many cell lines, LAK cell cytotoxicity to different tumor cell lines seemed to lx a suitable model for testing the new assay. The cytotoxicity values obtained using the MUH and MTT methods with adherent cell lines as the target are shown in Fig. 1. Both B16/Fl and B16FlO melanoma cells as well as Hep2 human epithelioma cells were efficiently killed by LAK cells. Increasing the number of effector cells caused increasing level of cytotoxicity. Using B16/Fl melanoma targets in 4 h MUH assays the percentage cytotoxicity values at ET ratios of 5~1, lo:l, 20:: and 50~1 were 14.10(~1.06), 26.43(+0.79),46.09(+6.44)and68.28(f5.64), respectively. The parallel MTF assay gave similar results: 10.29(+7.22), 22.Wf2.45). 46.81(+6.8) and

61.27(*

12.291,

respectively

(r = 0.991,

p <

0.00. When B16/FlO melanoma targets were used in 4 h MUH and MTT assays over the same range of ET ratios the following values were

obtained: 22.40(? 0.44). 39.12(f 3.01). 78.9% f 2.65) and 78.67( f 2.70) in the MUH assay; 22.02(*5.27), 30.14(+1.36), 69.4Nf2.53) and 76.40(*4.85) in the MYT assay (r = 0.987, p < 0.02). Beiig xenogenic to mice effwtors, Hep2 human epithelioma cells were more intensively killed by mouse LAK fells. Over the same range of ET ratios the followineI_nercentaee __cvtotoxiciw values wcrc obtained in MUH assays: 37.0X+2.03), 56.74(+0.30), 82.48(?1.97) and 101.59( f 2.99). The same culture terminated with MTF gave the following results 33.04(+10.05), 52.31(+7.16), 85.07(+6.11) and 101.93(+3.47), respectively (r = 0.998, p < 0.01). Summarising the results obtained from expcrimats with adherent targets, we can conclude that bath MIJH and W assays gave quantitatively wry similar results with close correlation over a substantial range of ET ratios. The MUH assay, however, gave more accurate results in terms of lower standard deviations. The two assays were also compared using non-adherent or partially adherent cell lines. Cytotoxicity against this type of cell cannot be measured with the previously described MUH method Wiigcr-Krasagakes et al., 1992). Our data demonstrate that this new assay is also able to measure cytotoxicity to non-adherent target cells (Fig. 2). Using a panel of weakly or non-adherent

1

MUH assay were higher. Percentage cytotoxicity values measured at the above E:T ratios with I&v5147 targets in the MUH assay were 11.21(~0.88), 25.74k1.891, 31.1EK~1.76) and 81.14(f6.39) in contrast to values measured with Ml-I-of 3.76(&2.13), 12.3Xf3.79), 27.52Ck2.15) and 61.3Xf7.01). respectively (r = 0.984, p < 0.02). Similarly, killing of Ta3St targets by LAK cells at the same effector:target ratios gave the following results with the MUH assay: 15.31(+_0.64), 27.93( f 1.23), 39.6X? 1.75) and 71.49(rt5.11) whereas wi:h the M’IT assay the values were 13.4Xf 2.39). 20.1SCf 2.17). 33.8% f 1.50) and 59.43Cf3.62). respectively (I = 0.996, p < 0.01). When Ta3Ha cells were used BS the target in 16 h cytotoxicity cultures at ET ratios of 1:2, l:l, 2:1, 5:l and l&l, the measured cytotocicity values in the MUH assay were 16.61(+0.91), 25.37Czt 1.25). 46.38(f0.75), 76.94( 2 0.51) and 95.08( f .5x%), respectively, whereas with MTT we obtained values of 9.45Ck6.991, 13.01Cf 1.38) 26.39Cf 1.671, 42.97tt4.94) and 68.31(f2.0!?) (r = 0.981, p < 0.01). 3.3. Reproducibility ofMUH cyfomxicity assay In order to assess the reproducibility of the new assay, IAK cell CT to 410.4 ceils were deter-

mined at ET ratios of 2~1, 5x1. 10~1 and 2O:l in 16 h assayx The same experiment was carried out on three different occasions. As shown in Fig. 3. the results could be reproduced with high accw racy. Differences of Cf values at no ET ratio exceeded 10%.

Since the use of the MUH assay for determining cell proliferation has already been described, setting up a compxhensive panel of experiments, as has been presented for the cytotoxicity assays, did not seem to be necessary. In order to provide further data supporting the suitability of the assay for determining cell proliferation, two simple models were used. Firstly, the growth-promoting effect of EIASN on the IM-dependent CXLL cells was determined (Fig. 4a) by bath methods. In this extensively used IL-2 bioassay very similar results were obtained showing close correlation between the hvo methods compared (r = 0.970; p < 0.01). In addition to this cytokine assay, another simple proliferation model waz established. Since all tumor cells gmwing in vitro need fetal calf serum in culture medium, the effect of different amounts of FCS on the proliferation of 410.4 adenocarcinoma cells was determined. As shown in Fig. 4b. both methods yielded very similar results with a high degree of correlation (r = 0.974, p < 0.003).

4. Discussion Radioactive chromium (%Zr) release to measure CMC and [3Hlthymidine incorporatirrn to measure cell proliferation are still the most widely used methods for the measurement of these basic immunological functions. The increasing awareness of environmental toxicity related to the widespread use of radioisotopes has led to the development of non-radioactive techniques. In most attempts fluorescent dyes have been used for this purpose. taking advantage of the high sensitivity that fluorescent techniques can provide.

The aim of this study was to test the suitability of 4-methylumklliferyl heptanoate for determining cell-mediated cytotoxicity and cell proliferation. The fluorogenic substrate is cleaved by intracellular esterases of living cells, as in the widely accepfed calorimetric MIT assay, in which a colorless tetrazolium salt is converted by mitochondrial dehydrogenases to blue forrrazan. On the understanding that both methods measure metabolic functions of liking cells, a comparison was made between the MUH and MTT methods for evaluating LAK cell cytotoxicity to different tumor cell lines, as a model of cell-mediated cytotoxicity. It is a conmum observation that different methods often yield considerably different percentage CT values (Parish and Miillbacher, 1983; Brenan and Parish, 1988) and some fluorescent CT methods give higher CT values compared with conventional techniques (Kriiger-Krasagakes et al.. 1992). We have found that without anv previous optimisation, quantitatively very similar results were obtained in both assays with many of the target cell lines. Although it seems to be a general finding that the new MUH assay yields higher values, a very close correlation was found between the two methods with the cell lines tested (Table 2). The fact that percentage CT values obtained by the new assay coincide with the data of the MTT assay supports the idea that a difference in assay principle (e.g., inhibition of metabolic functions vs. loss of membrane integrity) may he respxonsible for the different CT

values rather than a difference in detection technirlues (radioisotopic, lluorimetric or colorimetric). The most important advantage of our method is 1~sropidify. The romplete assay procedure from thl preparation

of cell suspensions until the corn-

pleuon of the measurement takes less than 5 h. This is due to the fast uptake and cleavage of the fluorogenic substrate by living cells (30 min), whereas the same process takes 4 h with tetrazolium salts in the MlT assay and an additional 30 min for the solubiliiation of the cleavage product. Another say is its trifugation

advantageous feature of the new asto the avoidance of cenwashing steps. This could be

simplicity due and

achieved by using modified colourless RPM1 1640 medium without phenol red in order to prevent high background fluorescence. Avoidance of washing may also contribute to the high accuracy. Finally, the new method can be applied to both adherent and non-adherent tumor cell lines. In all probability this simple assay protocol could also be automaied. Our data provide further proof of the use of the MUH proliferation assay and confirm its advantage.. Since the 48 h pmliferation cultures require the presence of FCS in the culture medium and fluorescence cannot be measured ic FCS-containing

medium

due to the high back-

ground caused by FCS, MUH

proliferation

assays

cannot be performed as simply as cytotoxicity assays (without centrifugation and washing). However, the accuracy of the new method and especially its rapidity (45 miu in contrast to the 290 min required for the MIT potential

assay) contirm the

value of the fiuorimetric

assay in this

field also.

Ackmwledgements The authors would like to thank Mrs. Gergely for excellent technical assistance.

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