Journal of Immunological Methods, 9 (1975) 7--26 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

AN IMPROVED FLUORESCENCE PROBE CYTOTOXICITY A S S A Y

R.J. BRAWN, C.R. BARKER*, A.D. OESTERLE, R.J. KELLY and W.B. D A N D LI K ER

Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037, U.S.A. *Department of HaematologicaI Medicine, University of Cambridge, Cambridge CB2 20L, England (Received 7 February 1975, accepted 20 May 1975)

The phenanthridine dye, ethidium bromide, which is actively excluded by viable cells, undergoes a significant fluorescence enhancement at 5900 A upon binding intracellular double-stranded polyribonucleotides. A rapid and sensitive assay of antibody mediated cytotoxicity to cells grown in vitro has been developed using this phenomenon. In this communication, we describe this fluorescence probe cytotoxicity assay and a sensitive electro-optical system designed to measure the fluorescence enhancement of ethidium bromide as it intercalates with intracellular polyribonucleotides. Basic characteristics of the fluorescence enhancement resulting from the interaction of ethidium bromide and non-viable cells are presented as well as examples of this assay as it has been used to study surface membrane neoantigens of cells transformed by the oncogenic DNA virus, SV40.

INTRODUCTION

Fluorescence probes are compounds which markedly alter their fluorescence quantum efficiency after binding an appropriate substrate. A large number of such probes are known and have been used to study enzyme, antibody and membrane structure (Winkler, 1962; Dandliker et al., 1964; McClure and Edelman, 1967; Stryer, 1968), as well as chromosomal structure (Latt, 1973). Fluorescence probes may also be quite useful as indicators of cellular damage. Le Pecq and Paoletti (1966) found that the phenanthridine dye, ethidium bromide (EB), undergoes a 20- to 25-fold fluorescence enhancement upon binding nucleic acids. They suggested that ethidium bromide fluorescence is specific for binding double-stranded regions of polyribonucleotides and developed a sensitive assay for ribonucleic acids using this fluorescence probe. Burns (1972) subsequently found that the fluorescence characteristics of ethidium bromide in the living cell are typical of RNA--EB complexes. DNA was felt to complex only a small amount of ethidium bromide in intact eukaryotic or prokaryotic cells. Edidin discovered that ethidium bromide is excluded from viable lymphoid cells but rapidly enters damaged cells and, in 1970, he described an in vitro cytotoxicity assay using this fluorescence Supported by PHS Research Grant No. RO5 CAl1650-03.

probe as an indicator of cellular damage. Edidin's fluorescence probe cytotoxicity assay had several advantages over other c o m m o n l y used antibodymediated c y t o t o x i c i t y assays. It was technically simpler and faster than most other assays. Furthermore, it had a sensitive and objective readout w i t h o u t the necessity of working with potentially dangerous radioisotopes. We have adapted this assay for use with target cells grown as monolayers in vitro and have designed an electro-optical system for detection of ethidium bromide fluorescence enhancement with greater range and sensitivity than previously available. In this communication we describe this modification of Edidin's fluorescence probe c y t o t o x i c i t y assay, as well as the electrooptical system designed to measure the 5900 A fluorescence enhancement of ethidium bromide as it binds intracellular polyribonucleotides. Basic characteristics of the fluorescence enhancement resulting from the interaction of ethidium bromide with non-viable cells in this system are presented and the sensitivity of the assay is compared to a Trypan Blue dye exclusion test. Examples of this assay as it has been used to study surface membrane neoantigens of cells transformed by the oncogenic DNA virus, SV40, are also presented. MATERIALS AND METHODS

Animals Inbred hamsters (LHC/LAK) were obtained from Lakeview Hamster Colony (Newfield, N.J.). In the experiments described, only male animals were used.

Buffers 1. Alsever's solution (pH 6.1): glucose (2.05%), Na3citrate • 2 H 2 0 (0.8%), citric acid (0.055%), and NaC1 (0.42%) were dissolved in distilled water and sterilized by autoclaving. 2. Membrane buffer 1 (MB-1): NaC1 (0.584%), ascorbic acid (0.35%), EDTA (0.74%), Tris--acid (0.118%) and Tris--base (0.03%), Na azide (0.01%), iodoacetamide (9.24%), and methane sulfonyl fluoride (0.0059%) were dissolved in distilled water and the pH adjusted to 7.6 with 1 M NaOH. 3. Membrane buffer 2 (MB-2): NaC1 (0.584%), ascorbic acid (0.35%), EDTA (0.74%), Tris--acid (0.118%) and Tris--base (0.03%) were dissolved in distilled water and pH adjusted as in MB-1. 4. Neutral buffered formalin: Na2HPO4 (7.8%), NaH2PO4 (4.2%) and excess solid MgCO 3 were mixed in 40% formaldehyde (aq.). This stock solution was diluted 10-fold with 0.85% NaC1 before use. 5. Phosphate-buffered saline (PBS): NaC1 (0.8%), KC1 (.02%), Na2HPO4 (0.115%) and KH2PO4 (0.02%) were dissolved in distilled water and pH adjusted to 7.4 with 1 M NaOH of 1 M HC1.

6. Tris--saline (pH 7.2): (0.015%) were dissolved 7. Tris--saline (pH 7.6): (0.030%) were dissolved

NaC1 (0.876%), Tris--acid (0.138%), and Tris--base in distilled water and sterile filtered. NaC1 (0.876%), Tris--acid (0.119%), and Tris--base in distilled water and sterile filtered.

Immunoglobulin (Ig) Hamsters bearing progressively growing SV40 induced sarcomas were bled by cardiac puncture and sera separated b y centrifugation after clotting at room temperature. Immunoglobulin was precipitated with ammonium sulfate to 40% saturation, re-dissolved in Tris--saline, pH 7.6, and the ammonium sulfate removed by Sephadex.G-25 gel filtration. The protein peak was sterile filtered through 0.45 p Millipore filter and stored in aliquots at --20°C. This immunoglobulin preparation was designated 106 : 192 and had a concentration of 7.9 mg/ml. Immunoglobulin preparation 106:192 was exhaustively absorbed with non-transformed cell membrane preparations in some experiments. Eightyone mg of syngeneic non-transformed cell membranes in Tris--saline, pH 7.6, were placed in 3 polycarbonate ultracentrifuge tubes and the membranes sedimented ( 1 0 0 , 0 0 0 g , 60 min, 4°C). The supernatants were discarded and 2 ml of immunog]obulin preparation 106 : 192 was added to one of the tubes, while the others were kept on ice. The membranes were suspended using a syringe and a blunt-tipped 18 gauge needle and mixed by continuous rotation for 30 min at room temperature. The membranes were removed b y sedimentation ( 1 0 0 , 0 0 0 g , 60 min, 4°C), and the supernatant containing the immunoglobulin carefully removed. The supernatant was sequentially absorbed with the t w o remaining membrane samples. The mg membrane/mg immunoglobulin ratio for each absorption was 5.1 : 1. This absorbed immunoglobulin preparation was designated 118 : 15.

Complement Rabbit c o m p l e m e n t free of anti-hamster or anti-SV40 heterophile antib o d y was prepared b y sequentially absorbing male rabbit serum on packed normal hamster red blood cells, formalin-fixed monolayers of the target cells, and, finally, freshly trypsinized target cells prior to each assay. Rabbit blood obtained from a large ear vein was allowed to clot for 30 min at room temperature and then for 60 min at 4 ° C. The serum was separated b y centrifugation (1250 g, 20 min, 4°C) and EDTA was added to 0.01 M. To prepare packed hamster red cells, several hamsters were bled b y cardiac puncture into a syringe containing 3 ml Alsever's solution. Three hamsters produced a b o u t 4.5 ml of blood which contained enough erythrocytes for the absorption of 40 ml rabbit serum. The cells were washed three times with 40 ml portions of cold 0.15 M NaC1. Half the hamster erythrocytes were incubated with serum from one rabbit for 15 rain at 4°C with intermittent mixing. The

10 red cells were removed b y centrifugation and the serum was reincubated under the same conditions with the remaining red cells. Two ml aliquots of this serum were then absorbed sequentially on t w o 85 mm diameter formalin fixed monolayers of line 506 SV40 transformed cells at 4°C and 60 min per absorption. The complement was stored in 1.5 ml aliquots at --70°C. For each assay, an aliquot of complement was thawed, diluted 1 : 1 with PBS, and incubated 30 min at room temperature with 106 trypsinized target cells. After removal of cells by sedimentation (500 g, 5 min, room temperature), it was mixed with 0.05 volumes of 1 M CaC12 and sterile filtered. Inactivated c o m p l e m e n t was used as a control for active complement in each experiment. Complement was inactivated by heating at 56°C for 30 min.

Target cells The target cells used in these experiments were derived from a transplantable SV40 induced sarcoma which developed in a LHC/LAK strain male hamster subsequent to a neonatal intramuscular injection of a 107 PFU/ml suspension of SV40 virus (obtained from Dr. K. Habel). This t u m o r line contains T antigen (Pope and Rowe, 1964) and S antigen (Tevethia et al., 1965) as determined b y indirect immunofluorescence. It has also been shown to express a transplantation antigen by viable cell challenge methods (Baldwin and Barker, 1967) subsequent to immunization with irradiated tissue. This t u m o r line was designated 506. In vitro cell cultures were initiated by enzymatically separating t u m o r tissue which had been surgically removed from tumor-bearing animals using aseptic technique. A crude suspension of t u m o r tissue was incubated with 0.05% trypsin plus 0.01 M EDTA in Eagle's MEM for 30 min. Cells were grown as monolayers in 85 × 20 mm sterile plastic petri dishes (Falcon Plastics, Oxnard, Calif.) in a humidified CO2 atmosphere at 37°C. Standard tissue culture medium consisted of Waymouth's Medium (Grand Island Biological Co., Oakland, Calif.) with 10% fetal bovine serum {Flow Laboratories), 1% MEM non-essential amino acid solution (Flow Laboratories), 1% 200 mM L-glutamine solution (GIBCO), 100 units penicillin/ml, and 100 pg streptomycin/ml brought to pH 7.2 with 7.7% NaHCO s. Cultured cells were brought into single cell suspension by exposure to 0.05% trypsin for 60 sec followed by inactivation with 0.01% ovomucoid (General Biochemicals, Chagrin Falls, Ohio) in 0.5% BSA. These cells were regularly assayed for mycoplasma contamination by plating tissue culture supernatants on PPLO agar. Mycoplasma contamination was not detected.

Mere brane preparation F o r t y f o u r grams of pooled kidney, liver, heart, lung and pectoralis muscle tissue was obtained from several normal male L H C / L A K strain hamsters using aseptic surgical technique. Immediately thereafter, the tissue was ho-

11 mogenized in a Waring blender in 60 ml of membrane buffer I (MB-1) until smooth. The homogenate was sedimented (600 g, 10 min, 4°C) to remove nuclei and whole cells. The supernatant was removed and the pellet was washed twice in MB-1. All supernatants were then pooled, brought to 300 ml with MB-1 and sedimented (600 g, 10 min, 4°C) to remove remaining nuclei or whole cells. The resulting supernatant was carefully removed and membranes were sedimented ( 1 0 5 , 0 0 0 g , 60 min, 4°C). The supernatant was decanted and lipid removed from walls of the centrifuge tubes with cottontipped swabs. The pellets were resuspended in membrane buffer 2 (MB-2) with a 20 cc syringe and long, blunt-ended 15 gauge needle, and again sedimented for 60 min at 100,000 g. After decanting the supernatant, the pellets were resuspended in Tris--saline, pH 7.6, sedimented (105,000 g, 60 min, 4°C), and resuspended in Tris--saline, pH 7.6, to a concentration of 1 gram of wet organ weight per ml. This suspension was distributed into 0.5 ml aliquots, snap-frozen in an ethanol/dry ice bath, and stored a t - - 7 0 ° C . Dry weight of these membranes in Tris--saline, pH 7.6, was 55.5 mg/ml.

Fluorescence probe cytotoxicity assay The supernatants from cultured target cells growing as monolayers at near confluency in 85 X 20 mm petri dishes were discarded and cells were brought into single cell suspension by exposure to 3 ml of 0.05% trypsin in MEM for 60 sec at room temperature. Cells were gently pipetted off the plate, put into a sterile conical centrifuge tube containing 3 ml of 0.01% ovomucoid trypsin inhibitor plus 0.5% BSA, and centrifuged 3 min at 500 g and the supernatant discarded. They were washed twice in 3 ml MEM plus 2% BSA and the cell concentration and viability determined visually after adding 20 pl of a 0.4% Trypan Blue dye solution to a 40 gl of the cell suspension. Trypan Blue excluding cells and Trypan Blue stained cells were counted using a standard h e m o c y t o m e t e r . Cell viability was usually 95--99%. The cell suspension was then diluted to 4.2 × 106 cells per ml using MEM plus 2% BSA, and 25 pl of this suspension was distributed to multiple 6 X 50 glass culture tubes (Owens-Illinois, Vineland, N.J.) using a 25 pl micropipette. F o r t y pl of immunoglobulin samples to be tested were added to the culture tubes which were placed on ice for 60 min with intermittent mixing using a Vortex mixer. Three hundred pl of MEM plus 2% BSA was added to each glass culture tube, and, after mixing, the cells were sedimented for 3 min at 500 g. The supernatant was withdrawn from each tube using a syringe and specially-adapted needle. This washing procedure was repeated once. Fifty pl of rabbit complement (active or inactive) was distributed to each of the glass tubes using a micropipette and the tubes were incubated on ice with intermittent mixing for 60 min. The cells were then incubated with intermittent mixing at 37°C for 60 min. Three hundred pl of a 4 pg/ml solution of ethidium bromide in MEM was added to each sample and, after a 15 min incubation at 37°C, the cells were added to a washed cuvette, diluted

12 with 3 ml PBS and the fluorescence of the ethidium bromide determined using the fluorometer to be described. Complement dependent cytotoxicity was determined by measuring the difference in fluorescence between samples incubated with active and inactive complement. Injured cells fail to exclude ethidium bromide, which, u p o n intercalating double-stranded cytoplasmic and nuclear polyribonucleotides, undergoes a marked fluorescence enhancement at 5900 A. Thus, increasing fluorescence at 5900 A indicates increasing cytotoxic effect. We have found it most convenient to record results in terms of arbitrary fluorescence units with the fluorescence of a glass reference standard and the fluorescence caused b y the c o m p l e m e n t dependent c y t o t o x i c i t y of a reference immunoglobulin preparation used to standardize assay readings from experiment to experiment. Calculation of complement dependent c y t o t o x i c effect in terms of per cent cytotoxicity, rather than arbitrary fluorescence units, can be easily accomplished by including in each assay an immunoglobulin preparation which is known to cause 100% cytotoxicity as determined by Trypan Blue dye exclusion assay. The formula for calculation of complement dependent cytotoxicity of each sample is then: Fluorescence % cytotoxicity = (100) (active complement) Fluorescence (100% cytoxicity )

Fluorescence (inactive complement) Fluorescence (inactive complement)

One hundred per cent cytotoxicity can also be achieved by multiple rapid freeze-thaws of a sample. However, the fluorescence of samples which have undergone multiple freeze-thaws is invariably greater than samples in which 100% c y t o t o x i c i t y has been achieved b y complement dependent antibody effect. This is probably due to the much greater interruption of membrane continuity caused by freeze-thawing as compared to antibody--complement reactions, and so we have chosen not to use fluorescence of freeze-thaw killed samples in the computation of cytotoxicity. Fluorometer

The optical and electronic schematics of the fluorometer designed for the fluorescence probe cytotoxicity assay are present in figs. 2, 3 and 4. The light source of the instrument is a Tungsten ribbon filament lamp (General Electric No. 18A/T10/1P-6V), powered b y a stabilized DC voltage source (Sorensen Nobatron QSB6-30), variable from 5 to 9 V. The use of a ribbon filament directly provides an image of the correct size and shape and makes it unnecessary to have multiple slits and diaphragms in the optical system. A heat-absorbing filter (Corning 1-69), an excitation interference filter peaked at 520 nm, and a quartz lens, which focusses an image of the light source into the sample cuvette are placed in the light beam. A quartz glass plate,

13

inclined at 45 ° to the incident beam, acts as a beamsplitter with the light reflected from the quartz plate going to a reference p h o t o t u b e (General Electric No. 935). The incident beam n o t reflected then enters the sample cuvette. Light emission from the cuvette passes through a barrier filter (Coming 3-66) which absorbs the 5200 A wavelength incident light and a lens which focusses the emission beam near the p h o t o c a t h o d e of a sensitive photomultiplier (EMI 9502S) supplied b y a stabilized high voltage (typically 1850 V) source (Fluke Model 412B). The 5900 £ fluorescence of the sample can be measured by potentiometrically determining the ratio of the photoc u r r e n t , i F , from the photomultiplier to that of the reference p h o t o t u b e , ix. Alternatively, the signal from each p h o t o t u b e can be electronically processed and a ratio, proportional to the ratio of the intensities of light impinging on the reference p h o t o t u b e and the photomultiplier, can be displayed digitally. In this case, voltages from the currents ii and i F are amplified by a follower module utilizing dual field-effect transistor inputs which reduce input current requirements to less than 50 pA, while maintaining amplifier o u t p u t accuracy at better than +0.0008 V over an input voltage range of + 10 V and an ambient temperature of 10--30 ° C. The follower module outputs, v1 and VF, go to a gain module which makes voltage levels and polarities compatible with the digital readout unit and increases the range of measurements possible w i t h o u t having to adjust other instrument parameters, such as lamp voltage or photomultiplier p o w e r supply. The module consists of t w o amplifiers, one inverting, one non-inverting, and a mechanism to switch the gain of both amplifiers simultaneously. The available gains are 1, 3, and 10, with an accuracy of 0.1%. The voltage levels from the gain module, e1 and eF, are measured by a DIGILIN Model 2330 digital panel meter in the ratio-measurement mode. The display module allows display of eF, el, eF/ei, or 5 X eF/ei. The ei voltage level is also compared to a 5 V internal reference by a comparator module. A sample readout cycle is begun by depressing a control module start switch which zeroes the follower module amplifiers for 0.1 sec. The control module then allows the follower module to monitor the charge accumulating on capacitors C, and C2. The acquisition of a 5 V signal for e~ (gain module output) causes the control module to p u t the follower module on " h o l d " for 30 seconds, during which time eF, ei, e F/ei o r 5 X e F/e I c a n be displayed on the digital output. At the end of 30 sec, the follower module is automatically zeroed. A new readout cycle may be initiated any time during or after the 30 sec 'hold' interval. RESULTS

In this paper, we (1) describe a successful modification of Edidin's fluorescence probe c y t o t o x i c i t y assay; (2) describe a fiuorometer with wider range and sensitivity than previously available for such assays; (3) present some

14

basic characteristics of the fluorescence enhancement resulting from the interaction of ethidium bromide (EB) with non-viable cells in this system; (4) compare the sensitivity of this assay with the commonly-used Trypan Blue dye exclusion c y t o t o x i c i t y assay; and (5) present several examples of this fluorescence probe assay as it is routinely used in our laboratory. Figure 1 indicates the structure of the fluorescence probe, ethidium bromide (2,7-diamin.o-2-phenylphenanthridine-10-ethyl bromide), used in the assay described. Figure 2 is an optical schematic of the fluorometer designed to measure the 5900 h fluorescence enhancement of EB as it intercalates intracellular ribonucleotides. Figure 3 is a system block diagram of the signal processing system and fig. 4 is a complete electronic schematic of the fluorometer. Figures 5 and 6 demonstrate some basic characteristics of the 5900 h fluorescence enhancement which results when EB and non-viable cells are mixed. Figure 5 demonstrates the 5900 & fluorescence of various concentrations of freeze-thaw killed cells with and w i t h o u t the addition of EB to a concentration of 4 pg/ml. The intrinsic fluorescence of the medium (MEM, plus 2% BSA) w i t h o u t cells is 0.097 fluorescence units. The change in 5900 h fluorescence intensity when killed cells are added to medium alone is negligible. The addition of cells to a concentration of 4 X 10 s cells/ml changes the fluorescence intensity from 0.097 units to 0.119 units. The 5900 £ fluorescence intensity of 4 pg/ml EB in MEM, plus 2% BSA is 0.255 fluorescence units. The increase in 5900 h fluorescence is dramatic when killed cells are added to medium plus 4 pg/ml EB. In this case, addition of killed cells to a concentration of 3.5 X 10 s cells/ml increases the fluoresPI

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C2H5 Fig. 1. Structure of ethidium bromide (2,7-diamino-9-phenylphenanthridine-10-ethyl bromide). Fig. 2. Fluorometer optical layout. Dimensions are to scale. F1, heat absorbing filter (Corning 1-68). F2, interference filter peaked at about 520 nm. F3, barrier filter (Corning 3-66). I, light source (General Electric 18A/T10/1P-6V). L1, lens focussing light source into the sample cuvette. L2, lens focussing the cuvette emission to the plane of the photomultiplier photocathode. P1, reference phototube (General Electric No. 935). P2, photomultiplier (EMI 9502 S); ( - ) 5200 • wavelength light; ( ~ ' ~ ) 5900 A wavelength light. Q, quartz glass beam splitter. S, slit to remove extraneous light scattered from the elements of the optical system.

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cence from 0.295 units to 0.786 units. The increase in fluorescence is linearly proportional to the concentration of killed target cells. Le Pecq and Paoletti (1966) previously noted that the 5900 A fluorescence enhancement of ethidium bromide solutions is linear with respect to the concentration of nucleic acid at low EB concentrations. In fig. 6, changes in 5900 A fluorescence with changes in EB concentration in different media are demonstrated. The increase in 5900 A fluorescence intensity is linearly proportional to increasing EB concentration in MEM medium, plus 0.2% BSA medium. The relationship between the 5900 A fluorescence and EB concentration is more complex when non-viable cells are present. In this case, the rate of increase in fluorescence with increasing EB concentration falls off at higher EB concentrations. This effect may represent competition for intercalation sites on double-stranded ribonucleotides. Two other factors could be important. If m a n y dye molecules are adjacent to one another, it is possible that a kind of dimer or polymer formation could occur with a subsequent alteration in fluorescence properties. This kind of interaction is felt to take place when acridine dye molecules intercalate the helix of nucleic acids and is probably a manifestation of the well-known tendency of dye molecules to aggregate in solution (Dandliker and Portmann, 1971). Excimer formation is another possible explanation. Excimers are dimers which are stable only in the excited state and are known to account for some anomalies in the fluorescence properties of aromatic hydrocarbons at high concentrations (Hercules, 1966). Figures 7, 8 and 9 show experiments done to investigate the nature of surface antigenic determinants of SV40 transformed cells using the fluorescence probe cytotoxic assay. Figure 7 demonstrates the complement dependent cytotoxic effect on syngeneic SV40 transformed cells of an immunoglobulin preparation from pooled sera of SV40 tumor-bearing hamsters. This preparation was tested before (106 : 192) and after (118 : 15) exhaustive absorption with syngeneic non-transformed cell membranes. Each immunoglobulin concentration was tested in triplicate and all resulting fluorescence determinations have been plotted. This demonstrates the variability in the

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Fig. 4. Fluorometer electronic schematic. Components are as follow: Ci , C,, 2 /_IF 200 V capacitors. C3_7, C 12, ClS-20, 0.1 PF 50 V disc capacitors. Cs, Cie, 0.02 PF disc capacitors. Cg, Ci4, 0.01 I_IF disc capacitors Ci , , 25 FF 15 V electrolytic capacitor. Ci 3, 10 /_fF 15 V electrolytic capacitor. Di, D2, 2N3906 transistor used as a low leakage diode. Base as cathode, collector as anode. D3_,, lN914 silicon diodes. 11, LED panel indicator, 5 V. ICI, ICI, ICs-ia, type 741 Op Amp ICs, IC,, Signet& NE555V timer. IC4, 7404 TTL HEX inverter. ICs, IC,, 7440 TTL dual 4-input NAND gate. Qi, Q2, U234 siliconix dual matched FET. Qa, Q4, Silicon PNP switching transistor. R, , Rl, Ra, 1 Mn resistors. Rs, R,, 270 R, 5% l/4 W resistor. R,, R7, R4s, R49, 6.8 Ma, 5% l/4 W resistors. Ri a, Ri i, RsO, Rs i, 100 Ka RL075 resistors. R,h, R,,, Rat),

R 52, 10 k62 PC mount potentiometer. Rie, 4.7 MR, 5% l/4 W resistor. R,,, Rl,, 10 kR, 5% l/4 W resistors. Ris, 4.7 ka, 5% l/4 W resistor. Rig, Rza, R46, 6.8 fi, 5% l/4 W resistors. Rs2, R2s, Rz7, 3 kR, 5% l/4 W resistors. R23, 2.7 Ma, 5% l/4 W resistor. Rz4, 2.2 kR, 5% l/4 W resistor. Rze, 100 12, 5% l/4 W resistor. *Rgs, 3.6 k!J RL075 resistor. *Rzs, 4.7 kc1 RL075 resistor. *Rse, 8.4 kc2 RL075 resistor. *Rsl, 24 ka RL075 resistor. *Rs2, Rss, 7.5 kfi RL075 resistors. *Rs5, 4.3 k62 RL075 resistor. *R36, 7.36 kfi RL075 resistor. *Rs7, 12 ka RL075 resistor. *R3s, 24 kR RL075 resistor. *Rss, 10 kR RL075 resistor. *These resistance values must he carefully matched to assure the correct gain ratios. R4,, 7.5 kfi, 5% l/4 W resistor. Rq2, R44, 47 kn, 5% l/4 w resistors. R43, 15 kc2, 5% l/4 W resistor. Ra5,

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Power supply, +15 V 0.25 Amps; 5 V 1 Amp; Electrostatics, Inc. Model 300. Galvanometer, Rubicon with a sensitivity of 1 na/mm deflection, 10 K ~ critical damping resistance. Potentiometer for potentiometric mode measurements, Helipot 10 K ~ , 10 turn, 0.1% linearity

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rng Ig/ml Fig. 7. C o m p l e m e n t - d e p e n d e n t c y t o t o x i c effect of i m m u n o g l o b u l i n preparations 106 : 192 (0) and 118 : 15 (A) on cultured S V 4 0 - t r a n s f o r m e d cells. Preparation 106 : 192 was an unabsorbed 40% a m m o n i u m sulfate precipitated fraction of SV40 tumor-bearing hamster serum, while preparation 118 : 15 had subsequently been exhaustively absorbed with syngeneic n o n - t r a n s f o r m e d cell membranes. The average 5900 ~ fluorescence of multiple inactive c o m p l e m e n t control samples is represented by ( ).

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.325[ mg Ig/ml Fig. 8. Effect of variable periods of trypsin proteolysis on cultured SV40-transformed cells with regard to their susceptibility to c o m p l e m e n t - d e p e n d e n t c y t o t o x i c i t y of immunoglobulin preparation 118 : 15. Target cells were incubated 60 sec (m), 300 sec (0) and 1800 sec (A) with 0.05% trypsin at 37°C, pH 7.2. The average 5900 A fluorescence of multiple inactive c o m p l e m e n t control samples is represented by ( ).

19 5900 £ fluorescence of different samples treated identically and is a measure of the cumulative error introduced b y the sample handling necessary in this assay. A significant a m o u n t of antibody from SV40 tumor-bearing animals which, in vitro, is c y t o t o x i c to SV40 transformed cells, can be removed by absorption with non-transformed cell membranes. The absorbed antibody may be directed against a normal membrane antigen which is cryptically located in vivo. Alternatively, it is possible that antibody directed against a sex-linked isoantigen was present in the pooled immunoglobulin preparation due to the inadvertent inclusion of serum from a female animal. The absorbed immunoglobulin, however, detects only t u m o r specific membrane antigens of SV40 transformed cells. Figure 8 demonstrates the effect of variable periods of trypsin proteolysis on line 506 SV40 transformed target cells with regard to their susceptibility to c o m p l e m e n t dependent damage by antibody against t u m o r specific membrane neoantigens. In this experiment, in vitro cultures SV40 transformed target cells were exposed 6 0 , 3 0 0 and 1800 sec to 0.05% trypsin at 37°C, pH 7.2. Trypsin was inactivated b y the addition of ovomucoid trypsin inhibitor and the cells washed twice in MEM plus 2% BSA. The c o m p l e m e n t dependent c y t o t o x i c effect of immunoglobulin preparation 118 : 15 was then determined using the fluorescence probe cytotoxicity assay. No decrease in susceptibility to the c y t o t o x i c effect of this immunoglobulin preparation was observed as trypsin proteolysis of the transformed cells increased. This demonstrates that t u m o r specific antigenic degradation during trypsinization to remove target cells from a monolayer conformation is n o t an important variable in this system, and suggests that the t u m o r specific antigenic determinant being detected may be a non-protein substance. Figure 9 demonstrates the effect of prolonged in vitro culture on the expression of SV40 t u m o r specific membrane antigen. Aliquots of line 506 SV40 transformed target cells were frozen in 10% DMSO in MEM after passage 19 and stored at --70°C while others were maintained'in continuous culture through passage 51. After this period of time, approx. 120 days, the passage 19 cells were recultured and both cell cultures assayed for expression of t u m o r specific antigen using immunoglobulin preparation 118 : 15. This experiment demonstrates that the sumeptibility of line 506 target cells to antibody against t u m o r specific membrane antigen did n o t change with prolonged in vitro cell culture. In fig. 10, a comparison between the sensitivity of the fluorescence probe c y t o t o x i c i t y assay and a standard Trypan Blue dye exclusion cytotoxicity assay is presented. In this experiment, the complement dependent c y t o t o x i c effect of different concentrations of immunoglobulin preparation 118 : 15 was determined by both assays using triplicate samples. After the 5900 A fluorescence of each sample was determined, a Trypan Blue dye exclusion viability assay was performed using the same cells. From the relative immunoglobulin concentrations at which the value of 5900 A fluorescence or % non-dye excluding cells is decreasing maximally, it can be seen that these

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.42o 380

.340 .500 mg lg / m l

Fig. 9. Effect o f in vitro culture time on the expression of SV40 t u m o r specific antigen. Line 506 target cells were grown 19 passages (0) or 51 passages (A) in vitro and simultaneously assayed for expression of t u m o r specific antigen using i m m u n o g l o b u l i n preparation 118 : 15. The average 5 9 0 0 / ~ fluorescence of multiple inactive c o m p l e m e n t control samples is represented by ( ).

m

.600

' t9 0 8o

.550

7o _~ 6o

.500

.450

4o ~ 30 ~

.400

,500

t

20 ~

•35q

I0

,~2

o.~6

' 0.55 mg Ig/ml

' - .165

Fig. 10. Comparison b e t w e e n the sensitivity of the fluorescence probe c y t o t o x i c i t y assay and the T r y p a n Blue dye exclusion assay. Multiple samples were measured for both 5 9 0 0 / ~ fluorescence (0) and the percentage of cells not excluding Trypan Blue (A). The average 5900 A fluorescence of multiple inactive c o m p l e m e n t control samples is represented by ( - ).

inactive active

inactive active

0.165

0.082

0.00

inactive active

0.33

active inactive

inactive

active

inactive

active

0.66

1.32

.520, .535, .508 .332, .333, .342 .333 .324

.598, .621, .631

.651, .666, .665

.380,.371, .390 .323,.323, .334 .333 .324

.435,.435, .435

.562,.531, .517

.608,.568, .561 .317,.311, .315 .622,.546, .580

506 P51 Tryp 60s

506 P51 Tryp 60s .709, .686, .694 .325, . 302, .327 .683, .662, .681

118:15

106:192

Figure 7

506 P52 506 P52 506 P19 Tryp 300s Tryp 1800s Tryp 60s

118:15

Figure 9

.313 .312

.330 .305

.333 .324

.327,.340,.314, .337,.303,.319,.338

. 3 5 6 , . 3 8 5 , . 3 6 2 , .377 . 3 8 7 , . 3 5 3 , . 4 2 3

.396,.408,.421, .416,.420,.375,.470

.470,.480, .475, . 4 8 9 , . 5 0 2 , . 4 4 2 , .563

.503,.513, . 5 2 0 , 1 5 4 8 , . 5 0 1 , . 5 4 9 , . 6 0 0

.320,.312,.327, .315,.318,.307,.333

.499,.498,.315, .544,.321,.326,.602

506 P52 Tryp 60s

118:15

Figure 8

Fig. 11. Presentation of all numerical data obtained in experiments 7, 8 and 9.

Ig Prep Target cell

Ig conc Complement mg/ml

.380, .371, .390 .323, .323, .334 .333 .324

.435,.435, .435

.562,.531, .517

.608,.568, .561 .317,.311 .315 .622,.546, .580

506 P51 Tryp 60s

['-D

22 two assays are very similar in their sensitivity. This is not surprising in that both assays are dependent on the p h e n o m e n o n of failure of dye exclusion by non-viable cells. In the case of the Trypan Blue assay, the dye is Trypan Blue and the failure to exclude the dye is determined visually. In the case of the fluorescence probe assay, the dye is ethidium bromide and the failure to exclude the dye is determined fluorometrically. Finally, fig. 11 presents the raw data from experiments 7, 8 and 9. The purpose of this figure is to show the numerical data derived from this assay as it is routinely used. The variability in readings of samples done in duplicate or triplicate as well as in the inactive complement controls is demonstrated. As can be seen, inactive complement controls are not routinely performed for each active complement sample. I n the type of experiment presented, the average fluorescence of the inactive complement controls does not change appreciably even when inactive complement controls are read for each active complement sample. DISCUSSION Several different assays for antibody or toxin-mediated cell c y t o t o x i c i t y have been described and are widely used. In one type of assay (Gorer and O'Gorman, 1956), the cytotoxic effect is measured by the failure of damaged cells to exclude vital dyes such as Trypan Blue, erythrosin B, or eosin Y. The readout of such assays is by visual enumeration of cells which have or have not excluded the dye to a degree which is visually detectable. Such assays are laborious and the readout is subjective. They have, however, proved to be very useful in investigating m a n y problems in immunobiology (Slettenmark and Klein, 1962; Old et al., 1965; Wahren, 1966). In the colony inhibition (Hellstrom and Sjogren, 1965), or microcytotoxicity (Hellstrom and Hellstrom, 1971) assays, target cells are grown at low density in petri dishes or wells of microtest plates. After becoming attached, they are incubated with antibody or toxins and the number of cells or groups of cells which are attached and show no evidence of cytotoxic effect are determined by visual enumeration hours to days later. This type of assay is quite sensitive but it is time-consuming, subject to problems with bacterial contamination, and has a very subjective readout. Release of radioactive markers from target cells is frequently used for quantitative determination of cytotoxic effects. In general, target cells are incubated with one of several different radioisotopically labeled substances, including DNA {Klein and Perlmann, 1963), proteins (Perlmann and Broberger, 1963), 3~p-phosphate (Perlmann and Broberger, 1963) or 5 ~Cr-chromate (Wigzell, 1965; Holm and Perlmann, 1967; Brunner et al., 1968). The target cells are then incubated with antibody and complement or with toxins. The total radioactivity of the sample and that of the cell-free supernarant (or insoluble residue) is determined. The released (or retained) isotope, usually expressed as percei~tage of total radioactivity, represents a cumula-

23 tive measure of cytotoxic effect. Another variation of this assay uses inhibition of cellular incorporation of radioisotopically-labeled substances such as amino acids as a measure of cytotoxic effect (Williams and Granger, 1969). These assays can be quite sensitive and the readout is objective and quantitative. However, they do necessarily involve the continued use of potentially dangerous radioactive substances, as well as a pre-assay incubation step in which the radioisotopically-labeled marker is concentrated intracellularly. An interesting alternative to these assays are those in which fluorescent molecules are used as indicators of cell damage. One such assay is the fluorochromasia c y t o t o x i c i t y test. This assay (Bodmer et al., 1967; Watanabe et al., 1971) is based upon the finding by R o t m a n and Papermaster (1966) that free fluorescein, produced by intracellular esterase-mediated hydrolysis of fluorescein diacetate esters, is retained by intact cells, but released by damaged cells. The readout of this assay is by visual enumeration of fluorescent cells, and thus the assay is laborious and subjective. Further problems include the fact that the degree of esterase activity differs considerably with different cell lines and that complement is capable of hydrolyzing fluorescein diacetate esters. Another variation involves the use of fluorescence probes which are actively excluded from healthy cells, but which enter injured cells and undergo a fluorescence enhancement upon combining with some intracellular substrate. In 1970, Edidin described a cytotoxicity assay using the fluorescence probe ethidium bromide. This assay was used to determine anti-H-2 antibody-mediated cytotoxicity towards fresh lymphoid cells and the readout used a commercially available fluorometer. The advantages of the assay he described were rapidity, an objective readout, and the fact that no radioactive substances were needed. Drawbacks of the assay were a restricted fluorometer readout scale and large degrees of non-specific complement toxicity. The specific physico-chemical changes necessary for fluorescence enhancem e n t are often not clear in individual cases, but some general guiding principles are evident. Probably the oldest k n o w n example of fluorescence enhancement occurs when the strongly-absorbing, non-fluorescent triphenylmethane dyes are adsorbed onto a variety of solid surfaces. This effect was classically interpreted as being due to molecular planarity acquired on adsorption, in contrast to some degree of free rotation in the unadsorbed dye. The polarity of the medium surrounding the dye is also often of determining importance. In the case of the anilinonaphthalenes, large fluorescence enhancements occur either when t h e y are bound to proteins or when they are dissolved in media of low dielectric constant. An excellent study (Turner and Brand, 1968) was made of this effect. These workers found t h a t a single straight line described the results of all water-solvent systems studied if the fluorescence intensity was plotted versus the Kosower Z parameter, which is a measure of solvent polarity (Kosower, 1958). A third type of effect is probably important in the large quenching effect observed when fluorescein

24 binds to an antifluorescein antibody (Portmann et al., 1971). Here, the lowered polarity of the surroundings after binding may well lead to a decrease in ionization of the fluorescein molecule, which in free solution drastically lowers the fluorescence emission. In this paper, we have described a modified fluorescence probe cytotoxicity assay which is adapted for use with target cells grown as monolayers in vitro. A sensitive fluorometer with an expanded dynamic range has been designed and has increased the capability of this assay significantly. The fluorescence probe c y t o t o x i c i t y assay is quite similar in sensitivity to the Trypan Blue dye exclusion assay, although both of these assays are clearly much less sensitive than a recently described neutral red stain delocalization c y t o t o x i c i t y assay (Filman et al., 1975). We have used the fluorescence probe cytotoxicity assay to investigate surface neoantigens of cells transformed by the oncogenic DNA virus, SV40. A few of these experiments are graphically presented and raw data from each experiment is included. A detailed discussion of the significance of these experiments is b e y o n d the scope of this paper. They do, however, demonstrate some pertinent points with regard to the immunology of the SV40 t u m o r system. It is possible to obtain antibody directed against truly tumorspecific membrane neoantigens of SV40 transformed cells. The expression of the t u m o r specific membrane antigens is not demonstrably affected by extended periods of in vitro cell culture, nor is the antigen degraded by trypsin proteolysis. The fluorescence probe c y t o t o x i c i t y assay is technically uncomplicated, and the readout quite rapid. R e a d o u t time, as the assay is routinely used, is 20 sec per sample. The digital readout represents a ratio of the sample emission signal at 5900 A accumulated over an arbitrary period of time to a signal directly from the excitation beam accumulated over the same time interval. The readout time interval is determined by the time necessary for the sum of the reference p h o t o t u b e signals to reach a set value. As lamp intensity increases, the time for the reference p h o t o t u b e signal to reach this set value decreases and thus readout time can be easily varied by varying lamp intensity. Alternatively, the readout time can be adjusted by changing the gain control of the fluorometer gain module. The major sources of error affecting readout accuracy are: (1) intrinsic fluorometer instability, (2) possible photochemical reactions occurring in the sample before and during reading, and (3) sedimentation of cells during reading. Intrinsic variability of readings from the fluorometer described is small. The standard error of a large series of sequential readings of a glass reference standard was -+1%. To decrease error induced by possible photochemical reactions occurring prior to and during reading, each sample is kept in the dark from the time the ethidium bromide is added to the time of sample reading and the fluorescence intensity of each sample is determined only once. Sedimentation effects are minimized by using a short reading time.

25 T h e f l u o r e s c e n c e p r o b e c y t o t o x i c i t y assay described in this p a p e r is in regular use in o u r l a b o r a t o r y . We feel it has distinct advantages o f r a p i d i t y , sensitivity and s a f e t y over various o f t h e p r e s e n t l y available c y t o t o x i c i t y assays. H o p e f u l l y , the i n f o r m a t i o n p r o v i d e d herein will be o f use to clinical or research l a b o r a t o r i e s w h i c h c o u l d use a c y t o t o x i c i t y assay with these characteristics. ACKNOWLEDGEMENTS T h e a u t h o r s gratefully a c k n o w l e d g e the technical assistance o f A. Hicks, S. Saling and J. Holtz. T h e a u t h o r s w o u l d like to express special a p p r e c i a t i o n t o Dr. Dennis R. M u r a y a m a f o r helpful suggestions and discussions.

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26 Wahren, B., 1966, Exptl. Cell Res. 42, 230. Watanabe, T., Y. Yagi and D. Pressman, 1971, J. Immunol. 106, 1213 Wigzell, H., 1965, Transplantation 3, 423. Williams, T.W. and G.A. Granger, 1969, J. Immunol. 1 0 2 , 9 1 1 . Winkler, M., 1962, J. Mol. Biol. 4, 118.