Micronuclei and Comet Assay

I. INTRODUCTION
To evaluate genotoxic effects of chemical substances in vitro, various methods are available differing in their sensitivity, their practicability, and, finally, the genetic end points considered. The micronucleus assay is one of the methods used to detect chromosomal aberrations in proliferating cell culture systems (Fenech and Morley, 1985). Its advantages are sensitivity as well as uncomplicated realisation and evaluation of results. Because of its relevance and applicability for human cell systems, the cytokinese-block Micronucleus assay with human lymphocytes is described in detail (Fenech, 2000). Micronuclei represent chromosome fragments or whole chromosomes which are not incorporated into the main nuclei at mitosis and they consequently appear only in dividing eukaryotic cells. In order to score micronuclei exclusively in cells that have completed one nuclear division only, the cytokineseblock method is applied in this test version, which prevents the cytoplasmic division after nuclear division by use of cytochalasin B.

The comet assay, or single cell gel electrophoresis, is another widely used assay for identifying genetic damage such as DNA strand breaks. The alkaline version of the assay introduced by Singh et al. (1988) allows the identification of DNA single strand breaks in a very sensitive manner. In addition, there are further advantages, such as the relatively low cell number (<10,000 cells per slide), its ease of application, the applicability of nearly all human and other eukaryotic cells proliferating in vitro, and its time- and costsaving performance, which argue for the use of this assay. To summarize main test principles: agaroseembedded cells are lysed by the help of high salts and detergents; DNA unwinding is promoted by incubating in alkaline electrophoresis buffer (pH > 13); and single-stranded DNA is electrophoresed under alkaline conditions, allowing the DNA fragments to migrate to the anode. After neutralization of slides, the amount of DNA damage may be detected by use of a fluorescent dye such as ethidium bromide visualizing the migrated DNA as the so-called comet tail. The migration length (i.e., the tail length) depending on the DNA fragment size is the most commonly used parameter for quantifying DNA damage. Other metrics considered only by use of image analysis systems are the percentage of migrated DNA and the tail moment (Olive et al., 1990).

Exemplarily, three cell types are presented considering different species (human and rat cells), different culture types (suspension or monolayer culture), and diversity in metabolic activation capacity (normal human bronchial epithelial cells and human lymphocytes versus rat hepatocytes). Selected cell types are capable for genotoxicity testing of single substances and complex mixtures (Mfiller et al., 2002), and the presented test protocols may be transferred to any other eukaryotic cell type proliferating in vitro.

II. MATERIALS AND INSTRUMENTATION
RPMI 1640 instamed (T 121-01) is from Biochrom AG Seromed, as well as the trypan blue solution (L- 6323), phosphate-buffered saline (PBS, L-182-01), collagenase CLS II (C 2-22), fibronectin from human plasma (L 7117), and gentamycine (A-271-23).

Epith-o-ser (FM-56-L) and Leibovitz's L-15 (PM-23-S) are from cc-pro, as well as fetal calf serum (FCS, S-10-L), trypsin-EDTA (Z-26-M), and penicilline-streptomycine solution (Z-13-M). Minimum essential medium Eagle (MEM, M-1018) is from Sigma-Aldrich Chemie, as well as phytohaemagglutinine (PHA, L-9132), cytochalasine B (C-6762), CaCl₂ anhydride (C-1016), MgSO₄ × 7H₂O (M-1880), acridine orange (A-6014), phenol red (P 3532), insulin from bovine pancreas (I-6634), N-[tris(hydroxymethyl) methyl]-2-aminoetharesuforic acid (TES, T 5691), HEPES (H 4034), tricine (T 5816), MgCl₂ × 6H₂O (24,696-4), Na₂HPO₄ × 2H₂O (21,988-6), and Na₂HPO₄ × 7H₂O (22,199-6) and Na₂HPO₄ anhydride (S-9763). Normal melting temperative agarose (NMA, 11400) and collagen R (47254.02) are from Serva, as well as ethidium bromide (EtBr, 21251), glucose-6-phospate (22775.01), and Na-pyruvic acid (15220). Low melting temperature agarose (LMA, 35-2010) is from peqLab. Triton X-100 (6683.1), NaCl (3957.2), Na-EDTA (8043.1), Tris (4855.2), dimethyl sulfoxide (DMSO, 7029.1), ethanol (P.076.1), and isopropanole (6752.1) are from Roth. Lymphocyte separation medium (Ficoll-Paque, 17 1440-03) is from Amersham Biosciences. NADP (93208) and formic acid (33015) are from Fluka. Aroclor 1254 (RPC-1254) is provided by Ultra Scientific. All other chemicals are from VWR International: glucose anhydride (1.08337.1000), Na₂HCO₃ (1.063290.500), methanol (1.06009.1000), glacial acetic acid (1.00056.1000), NaOH (1.06462.1000), KH2PO₄ (1.04873.0250), methoxymagresium-methylcarborate (MMC, 8.18156.0100), KCl (1.04933.0500), Na₂SO₄ (106647), H₂O₂ (1.08597.1000), 2-mercaptoethanol (1.15433.0050), and L-glutamine (1.00289.0100). Heparin sodium salt (101931) is from ICN Biomedicals Inc. Bovine serum albumin fraction V (11018-025) is from Invitrogen. Peanut oil is from the pharmacy of the clinic of Martin Luther University Halle-Wittenberg.

For preparation of lymphocytes, Leucosep vials (227.290) are used, which are from Greiner. For Aroclor treatment of rats, a gauge needle (Sterican, 4667123) from B. Braun Melsungen AG is used. Surgery instruments are from Sigma Chemie: curved microdissecting forceps (F 4142), dressing tissue forceps (F 4267), straight microdissecting forceps (F 4017), curved microdissecting scissors with a sharp point (S 3271), straight microdissecting scissors with a sharp point (S 3146), scalpel blade (S 2771), and handle (S 2896). Cell culture supplies are from TPP: culture dishes (60cm², 93100), cell culture vials (20cm², 91106), cryo vials (2.0ml, 89020), 15-ml centrifuge vials (91015), 50-ml centrifuge vials (91050), serological pipettes (10ml, 94010), and culture flasks (25cm², 90026). Automatic pipettes (Eppendorf research variable: 0.5-10, 10-100, 20-200, 100-1000, and 500-5000 µl) are from Eppendorf. The pipette aid (Drummond pipette aid, 28081410) is from Heinemann Labortechnik.

Heating plate (453Nl120), water bath (Memmert WB 7, 4623520), swap thermostat (MP 5, 4613411), membrane pump for liquids (ND 100 KT.18, 2245110), electrophoresis chamber (Phero-Sub 2, 344797), power supply (Consors E835, 5822150), and the gel carrier (5817129) are from VWR International. The cell counting chamber (Fuchs-Rosenthal, T731.1), minicentrifuge (X409.1), staining tanks (Hellendahl, H549.1), slides (76 × 26 × 1 mm, 0656.1), and coverslips (24 × 40mm, 1870) are from Roth. For the comet assay, fully frosted slides (61224) from Menzel are used. The CO₂ incubation chamber CB 150 (9040-0001) is from wtb-Binder. Clean bench Uniflow (KR-125-GS) is from UniEquip. The table centrifuge (Megafuge, 75003060) is from Kendro. For microscopic analysis, a Nikon fluorescence microscope Eclipse E 600 (MBA 70400) is used. Data processing is performed with the Komet analysis system, including software Komet 4.0 from Kinetik Imaging Ltd., purchased by BFI Optials.

III. PROCEDURES
A. Preparation and Cultivation of Cells
All cell types are cultivated in a CO₂-incubation chamber under the same conditions: 37°C 5% CO₂, and 95% relative humidity.

1. Human Lymphocytes
Solutions:
Heat-inactivated FCS: Fill the necessary volume of FCS in a centrifuge vial, deposit the vial in a water bath, and heat the water continuously up to a temperature of 56°C. Hold this temperature for 30min.

RPMI 1640 cell culture medium: To prepare 1 litre of cell culture medium, add the specified volume of powdered RPMI 1640, 2g NaHCO₃, and 750ml dH₂O while stirring. Complete to 890ml with dH₂O, adjust pH to 7.4, and store at 4°C. Before use, add 100ml of heat-inactivated FCS and 10ml of penicilline-streptomycine and proof the pH to be 7.4.

Phosphate-buffered saline (PBS): To make 1 litre of PBS, add the specified volume of powdered PBS (Ca²⁺/Mg²⁺ free) and 750ml dH₂O while stirring. Complete to 1 litre with dH₂O and store at 4°C

Steps
Before starting cell preparation, warm up all media, buffer, and supplements in a water bath (37°C). Use certified buffy coat or other human blood preparation.

1. For setup of the vials, pour 15ml Ficoll-Paque in 50 ml Leucosep tubes and centrifuge at 900g for 2 min.
2. Mix cold buffy coat with warm PBS in 1:2 proportion.
3. Layer 30ml of this buffy coat PBS mixture onto a separation ring of the centrifuged Leucosep tubes and centrifuge at 800g for 25min, not using the brake.
4. After centrifugation, remove the lymphocytes carefully from the white lymphocyte ring, deliver them in a new tube, wash with a double quantity of PBS, and then centrifuge again at 800g for 10min.
5. Perform a second washing with 40ml PBS and repeat centrifugation (800 g for 10 min).
6. Finally, resuspend lymphocytes in warm RPMI 1640 culture medium and cultivate the cells overnight in cell culture vials to allow sedimentation of the mononuclear cells, B lymphocytes, and regeneration of lymphocytes from the isolation procedure.

2. Hepatocytes of Rat
Solutions
Presuspension buffer: To make 2 litres, add 1500ml dH₂O, 16.6 g NaCl, 1 g KCl, and 4.8 g HEPES while stirring. After dissolving, complete to 2 litres with dH₂O and adjust pH to 7.4. Keep at 4°C.

Collagenase buffer: To make 1 litre of stock solution, add 750ml dH₂O, 3.9 g NaCl, 0.5 g KCl, 0.7 g CaCl₂ × 2 H₂O, and 2.4 g HEPES while stirring. After dissolving, complete to 1 litre with dH₂O and adjust pH to 7.6. Keep at 4°C. To make 200ml of working solution, add 27,300 U of collagenase.

Suspension buffer: To make 1 litre, add 750ml dH₂O, 4g NaCl, 0.4g KCl, 0.13g MgCl₂ × 6 H₂O, 0.15g KH₂PO₄, 0.1 g Na₂SO4, 7.2g HEPES, 6.9g TES, and 6.5 g Tricine while stirring. After dissolving, adjust pH to 7.6. Furthermore, add I g glucose, 30 g BSA, and 5 mg phenol red and sterilize by filtration. Keep at 4°C.

Pentobarbital solution: To get 1 ml, weigh 50mg of pentobarbital and complete to 1 ml with dH₂O.

Heparin solution: To make 2.5ml, weigh 1 mg heparin sodium salt and complete to 2.5ml with 0.9% NaCl.

MEM culture medium with Hank's salts, L-gluthamine, and nonessential amino acids: To prepare 1 litre, add the specified volume of powdered MEM, 320mg NaHCO₃, and 750 ml dH₂O while stirring, complete to 1 litre, adjust pH to 7.6, and aliquot. Keep at 4°C. Before use, weigh 0.5µg/ml insulin from bovine pancreas and 50µg/ml gentamicin and add to medium.

Steps
Hepatocytes may be isolated from untreated young laboratory rats by the in situ perfusion method with collagenase buffer following the modified methods by Berry and Friend (1969) and Seglen (1976). Note: It needs a special authorisation and large experience to perform manipulations with laboratory animals. Therefore, the method described in the following is only a summary.

1. Sterilise all surgical instruments, several glass beakers, and tubes. Warm up presuspension buffer and collagenase buffer (water bath, 40°C)
2. Anaesthetize rat with ip injection of 5mg/100g body mass pentobarbital (0.1 ml/100 g body mass) and inject 1 ml heparin solution per 250g body mass through the penis vein.
3. Open the rat, dissect vena portae, and induct a gauge needle for injection the buffer solutions. Fix the needle by ligature.
4. Open the thorax, cut the superior caval vein, and start perfusion with warm presuspension buffer for 15-20min at a constant flow rate of 10ml/min.
5. Continue perfusion with warm collagenase buffer for about 10-20min until the liver loses its typical elasticity and colour and becomes soft and pale. (Among other things, perfusion time depends on the activity of collagenase, flow rate, and constitution of liver.)
6. Gently dissect liver lobes and transfer to a culture dish with cold suspension buffer.
7. Carefully remove liver capsule and mechanically disaggregate cells using two forceps. Filtrate the cell suspension through different layers of gauze, transfer suspension to centrifuge vials, and centrifuge at 50g for 6 min (4°C).
8. Discard supernatant and resuspend pellet in MEM medium. Transfer cell suspension to culture vials. Adjust cell number to 1 x 107 cells/ml after counting and estimation of viability by the trypan blue method (see later).
9. Use freshly prepared hepatocytes in tests without cultivation.

3. Normal Human Bronchial Epithelial Cells (NHBEC)
NHBEC may be isolated from explants from the bronchial airways of patients after lob- or pneumectomia (i.e., the resection of one of the lobes or of the lung at a hemithorax because of bronchial carcinoma) following the method of Lechner and LaVeck (1989) modified by Stock (2002).

Solutions
Epith-o-ser culture medium with supplements (serum free): Thaw the frozen supplements delivered and add to 500 ml of basal medium. Prepare the medium before use.

L-15 medium: To make 1 litre, dissolve the specified volume of powdered L-15 in 750ml dH₂O and complete to 1 litre with dH₂O.

Coating solution: To make 100 ml solution, dissolve 1 mg fibronectine, 3 mg collagen R, and 1 mg BSA in 90 ml L-15 medium while stirring. Complete to 100ml with L-15 and filtrate. Keep at 4°C. Trypsin-EDTA solution: Aliquot the delivered solution (0.05% trypsin/0.02% EDTA in Ca²⁺- and Mg²⁺-free PBS).

Steps

1. Coat culture dishes: Transfer 0.2ml/cm² coating solution into culture dishes (60mm diameter) while working under a clean bench. Incubate at 37°C, 5% CO₂, and 95% relative humidity for 24h. Aspirate remaining solution and let the layer dry. Dishes may be stored enveloped in folio at 4°C for about 6 weeks.
2. Fragment the bronchial tissue into pieces of 5 to 10 mm 2 and wash in PBS tree times.
3. Place six to eight tissue pieces into a coated culture dish (see earlier discussion), close, and allow adhesion for 5 min.
4. Add 4ml of epith-o-ser medium and cultivate cells while changing culture medium every 2-3 days until a subconfluent status (about 2 weeks).
5. Before use of cells in the comet assay, carefully remove culture medium from the well, add warm 1.5ml trypsin-EDTA solution, and allow digestion for about 5 min (37°C).
6. To stop digestion of cells, add 4.5 ml cold PBS and gently move the well in order to get all cells free from the well surface.
7. Transfer cell suspension into a centrifuge vial, centrifuge at 125g for 10min, remove the supernatant, and resuspend the pellet in fresh medium.
8. Calculate cell number and test viability by the trypan blue method (see later).

Viability Test by Trypan Blue Method
Dilute 1 ml of cell suspension with 9ml culture medium or buffer, mix gently, extract 1 ml of this mixture, and add 1 ml trypan blue solution. Identify living (pale) and nonliving (blue) cells.

B. Micronucleus Assay
Peripheral lymphocytes are one of the most applied cell types for the identification of micronuclei in human tissue. Therefore, the method of the cytokineseblock micronucleus assay with human lymphocytes is described (Fenech, 2000).

Solutions
RPM11640 cell culture medium: To prepare 1 litre of cell culture medium, add the specified volume of powdered RPMI 1640 as described in the catalogue to 750 ml dH₂O while stirring. Complete to 890 ml with dH₂O, adjust pH to 7.4, and store at 4°C. Before use, add 100 ml of FCS and 10 ml of penicilline-streptomycine and proof the pH to be 7.4.

Fixative: To make 100ml, add 75 ml of methanol and 25 ml of glacial acetic acid and keep at 4°C.

Staining solution: To make 100ml of stock solution (0.24 mM), take 100 µl acridine orange and complete to 100ml with dH₂O. Store at 4°C. To make 100ml of working solution, add 1 ml of stock solution and 15 ml of Soerensen buffer.

Soerensen buffer: 60 mM Na₂HPO4: 60 mM KH₂PO4 = 1:1, pH 6.8.

Solution 1: To make 1 litre, weigh 11.876 g of Na₂HPO₄ and complete to 1 litre with dH₂O.

Solution 2: To make 1 litre, weigh 9.078 g of KH₂PO₄ and complete to 1 litre with dH₂O.

To get 100ml of buffer with pH of 6.8, add 50ml of Solution 1 and 50ml of Solution 2.

Steps
Perform the micronucleus test following the modified methods of Fenech and Morley (1985) and Sgura et al. (1997).

1. Resuspend cultivated lymphocytes, centrifuge at 800g for 5min, and remove the medium above the pellet.
2. Resuspend the cells in fresh RPMI medium supplemented with 10% inactivated FCS and 1% penicilline-streptomycine-solution at a concentration of 1 × 10⁶ cells ml, add PHA in a final concentration of 2mg/ml, and cultivate for 20h.
3. After this time, add the test substance in the selected test concentrations. Cultivate cells for 24h.
4. Add cytochalasin B in the final concentration of 3µg/ml and continue cultivation for another 24h. (Cytochalasin B prevents cells from completing cytokinesis, resulting in the formation of multinucleated cells.)
5. Centrifuge this suspension at 425g for 10min, remove the supernatant, wash the pellet with 2 ml PBS, and centrifuge again at 425g for 10min.
6. After removing the supernatant, resuspend cells in 1 ml of fixative, centrifuge at 200g for 5 min, aspirate the supernatant, and dissolve the pellet in few drops of fixative.
7. Transfer the fixed cells to normal, precleaned airdried slides. Stain the cells with acridine orange solution for 3 min, rinse three times with Sörensen buffer, and finally cover with coverslips.
8. Score the slides (two slides per concentration and control) for micronucleated lymphocytes: 1000 binucleate lymphocytes have to be scored for the number of micronuclei using a fluorescent microscope (400× magnification) fitted with a epifluorescent condensor and filter set (excitation filter 510-590nm). Perform two replicate experiments for each test.
9. Statistical differences between controls and each treated sample may be identified with the one-tailed χ² test following Lovell et al. (1989).

C. Alkaline Single Cell Gel Electrophoresis (Comet Assay)
Solutions
Trypsin-EDTA solution

NMA in aqueous solution: To make 100ml 1% agarose solution, add 1 g NMA to 100ml distilled water, heat carefully until the agarose is dissolved, and store at 4°C.

NMA in PBS: To make 100ml of a 0.6% solution, add 0.6 g NMA to 100ml PBS (without Ca²⁺ and Mg²⁺), heat carefully until agarose is dissolved completely, and store at 4°C.

LMA in PBS: To make 100ml of a 0.5% solution, add 0.5g LMA to 100ml PBS (without Ca²⁺ and Mg²⁺), heat carefully until agarose is dissolved completely, and store at 4°C.

Lysing solution: To make I litre, add 1.21 g Tris (10 mM), 146. 1 g NaCl (2.5M), and 37g Na-EDTA (100mM) to about 700ml dH₂O while stirring. After dissolving, complete to 890 ml with dH₂O, adjust pH to 10, and keep at room temperature. Immediately before use, add 1% Triton-X and 10% DMSO.

Electrophoresis buffer: To make 1 litre, add 0.37g Na-EDTA (1 mM) and 12g NaOH (300mM) to about 700 ml dH₂O under stirring. After dissolving, complete to 1 litre with dH₂O and adjust pH to 13. Prepare fresh.

Neutralisation buffer: Per litre, add 48g Tris to 750ml dH₂O and complete to 1 litre with dH₂O (400mM, pH 7.4).

Ethidium bromide solution: To get 10ml, dissolve 200µg EtBr in distilled water and complete to 10ml. Aliquot in 1-ml portions and keep at 4°C.

Steps
1. Preparation and Treatment Schedule of Cells

1. Human lymphocytes and rat hepatocytes (or other cells in suspension culture): Decant medium from lymphocyte overnight culture or from hepatocyte suspension, respectively. Centrifuge cells for 5 min at 200g. Resuspend cells in fresh culture medium. Calculate cell number (Fuchs-Rosenthal counting chamber) and test viability by the trypan blue method (see earlier discussion).
2. In case of culture treatment with test substance (in vitro experiments): Decant medium from deposited cells, add mixture of culture medium, test substance in the required test concentration, resuspend cells in this mixture, and cultivate while stirring vials gently.
3. After treatment, centrifuge cells for 5rain at 200g, discard supernatant, resuspend pellet in fresh culture medium, decant medium from deposited cells, and resuspend cells gently in 85~tl melted LMA per slide.

NHBEC (or Other Monolayer Cultures)

Short-term Treatment (1-2 hs)

1. Carefully remove media from subconfluent cell culture, add 1.5 ml of warm trypsin-EDTA solution, and incubate for 5 rain.
2. Follow steps 6-8 as described in Section III,A,3.
3. Centrifuge cells for 5 min at 200g, discard supernatant, add mixture of culture medium and test substance in the required test concentration, resuspend cells in this mixture, and cultivate while stirring vials gently.
4. After treatment, centrifuge cells for 5 min at 200g, discard supernatant, resuspend pellet in fresh culture medium, decant medium from deposited cells, and resuspend cells gently in 85µl melted LMA per slide.

Long-term Treatment (>2 hs)

1. Carefully remove media from subconfluent cell culture, add mixture of culture medium, and test substance in the required test concentration. Cultivate cells over the required treatment period.
2. After treatment, remove the treatment solution carefully, rinse with fresh medium, remove it, and add 1.5ml of warm trypsin-EDTA solution. Incubate for 5 min.
3. Follow steps 6-8 as described in Section III,A,3.
4. Centrifuge cells for 5min at 200g, discard supernatant, resuspend pellet in fresh culture medium, decant medium from deposited cells, and resuspend cells gently in 85µl melted LMA per slide.

2. Preparation of Cells from in vivo Experiments
Follow the procedures described earlier except for the treatment of cells.

Cells from Suspension Culture. Decant medium from cultured cells and resuspend cells gently in 85 ],tl melted LMA per slide.

Cells from Monolayer Culture

1. Carefully remove media from subconfluent cell culture, add 1.5 ml of warm trypsin-EDTA solution, and incubate for 5 min.
2. Follow steps 6-8 as described in Section III,A,3.
3. Centrifuge cells for 5min at 200g, discard supernatant, resuspend pellet in fresh culture medium, decant medium from deposited cells, and resuspend cells gently in 85µl melted LMA per slide.

Note: For administration of LMA, do not exceed temperature of 37°C. Adjust cell number to 10,000 per slide. In human cell culture systems (lymphocytes and NHBEC), S9 mix as an external metabolising system should be involved (described in Section III,D) in parallel experiments. Do not treat cells longer than 3h with S9 mix.

3. Slide Preparation
The slide preparation follows the basic method of Singh et al. (1988). All steps following step 3 are performed in a dark room under yellow light.

1. Melt NMA (1% in dH₂O) in a microwave (1 ml per slide). Cover a labelled fully frosted microscopic slide with 1 ml of NMA, spread it evenly, and remove the layer after drying by scratching.
2. Overload the slide with 300µl of melted NMA (0.6% in PBS), cover with a large coverslip, and store at room temperature until the agarose is solidified.
3. Meanwhile, prepare cell suspension (see earlier discussion).
4. Remove coverslip gently and transfer the cell agarose suspension onto the prepared slide.
5. Cover with a fresh coverslip and incubate for 10 min on ice. After the agarose is solidified, slide off coverslip gently and apply a layer of 85 µl melted LMA.
6. Cover with a fresh coverslip and incubate for 10 min on ice.
7. Slide off coverslip gently and incubate slide in freshly prepared chilled lysing solution for 1h at 4°C in the dark using a staining tank.
   
   Note: Steps 5 and 6: For incubation of slides on ice, prepare an ice bath, cover it with a piece of blotting paper, and apply an appropriate pane of glass.

4. Electrophoresis

1. Remove slide from the lysing solution and wash gently with fresh electrophoresis buffer (pH > 13).
2. Apply slides side by side (without distances) on the horizontal gel box of the electrophoresis chamber, fill the chamber with freshly made electrophoresis buffer, avoiding bubbles, and incubate slides for 1 h in order to allow DNA unwinding. The buffer just has to cover the slides!
3. Switch on power supply: Conduct electrophoresis at 25 V and 300 mA (0.66 V/cm) for 30 min and cover the chamber with aluminium foil.
4. Switch off power supply, remove slides from the chamber gently, and rinse carefully with neutralisation buffer three times to remove alkali and detergents. It is possible to store slides in neutralisation buffer until examination in the dark at 4°C, but do not exceed 24 h.

5. Examination

1. Gently remove slides from neutralisation buffer, place on a slide drainer, and stain every slide after draining with 60µl EtBr. Cover with a coverslip and allow staining for 10 min.
2. For DNA analysis, use an epifluorescence microscope with a 510- to 590-nm excitation filter and a short arc mercury lamp. Examine at 400-fold magnification. Per test concentration not less than 50 cells should be scored; two replicate experiments should be performed per test. It is possible to determine comet tail length as a result of DNA damage by use of a micrometer in the microscope eyepiece. However, this method is time-consuming and is subject to various mistakes. A better method of processing data is to use an imaging analysis system in connection with special software for the comet assay, such as described earlier. In this case, follow the manual.
3. To determine the lowest test concentration at which a significant increase in DNA damage (given as comet tail length or tail moment) has occurred, multiple pairwise comparisons have to be conducted between control data and each dose using the Student's t test.
   
   Note: To avoid confusion of results by concomitant processes leading to apoptosis, do not observe highly damaged cells. To ensure reproducibility of data between various experiments, do not use short arc mercury lamps with different wattage.

D. Further Procedures
1. Preparation of S9 Mix from S9 Fraction
Most of the cell culture systems, apart from hepatocytes, should be exposed to test substance both in the presence and in the absence of an external metabolising system, i.e., the postmitochondrial supernatant of the rat hepatocyte preparation of Aroclor 1254 pretreated male rats ($9 mix) as described by Czygan et al. (1973), Ames et al. (1975), and Natarajan et al. (1976), to consider the effectivity of test substance in dependency on its metabolisation. Usually, the final concentration of $9 mix ranges from 1 to 10%, depending on the classification of test substance.

2. Preparation of S9 Fraction
Solutions

1. Aroclor 1254: Suspend 200 mg Aroclor in 1 ml peanut oil.
2. 0.15M KCl: Dissolve 5.59g KCl in destilled water and fill up to 500ml; sterilize this solution by filtration. Store at 4°C.

Steps

1. Aroclor is administered to rat by ip injection using a final dose of 500mg/kg. Because of the high viscosity of the Aroclor suspension, use a gauge needle not smaller than 18 gauge.
2. Five days after injection, anaesthetize the rat, open the abdomen, and bleed and remove the liver immediately while working under sterile conditions at 4°C.
3. Weigh the liver, cut it into small pieces, and mix with cold sterilized KCl (1 g per 3 ml KCl).
4. Homogenize this mixture and centrifuge at 6000g at 4°C. for 10min using sterile centrifuge vials.
5. Aliquot the supernatant to sterile cryo vials and store frozen at -80°C.

3. Preparation of S9 Mix
Solutions
Stock phosphate solution: (a) Dissolve 4.804 g KH₂PO₄ in dH₂O and complete to 100 ml. (b) Dissolve 6.286 g Na₂HPO4 in dH₂O and complete to 100ml. Mix 19.6 ml of solution a with 80.4 ml of solution b, adjust pH to 7.4, and sterilize by heat (20min at 121°C in autoclave). Store at 4°C.

MgCl₂: Dissolve 406.51 mg MgCl₂ × 6 H₂O in dH₂O and complete to 50 ml, aliquot, and sterilize by microfiltration. Store at 4°C.

KCl: Dissolve 615mg KCl in dH₂O and complete to 50ml and sterilize by microfiltration. Store at 4°C.

NADP: Dissolve 726 mg NADP in distilled water and complete to 20ml and sterilize by microfiltration. Aliquot and store frozen at -30°C.

Glucose-6-phosphate: Dissolve 304 mg glucose-6- phosphate in dH₂O and complete to 20ml, aliquot, and sterilize by microfiltration. Store at -30°C.

Steps

1. To thaw components slowly, put all vials and bottles in an ice bath.
2. To make 10 ml of S9 mix, take 3 ml of stock phosphate solution, add 2 ml of KCl solution, 2 ml of MgCl, and 1 ml (10%) of S9 fraction. Complete this mixture with 1 ml NADP solution and finally add 1 ml of glucose-6-phosphate solution. It is very important to follow this sequence exactly and to take care that all components are mixed completely before adding the next one.

IV. COMMENTS
In each experiment, in addition to the test concentrations, a negative control (culture medium) and a positive control should be included for validation of results [micronucleus test: 0.5M MMC, following Surral6s and Natarajan (1997), comet assay: 300µM H₂O₂]. If a test substance has to be dissolved or suspended in any vehicle, a vehicle control has to be included too.

It is important to determine the concentration range as well as the treatment period in prestudies for every test substance and for various cell systems. The number of different test concentrations should be three or more. For more detailed description of test parameters, see Tice et al. (2000).

V. PITFALLS

1. In the case of elevated room temperature (about 30°C or more) it is possible that LMP agarose will not solidify; therefore, keep the slides together with the ice bath into the refrigerator.
2. To assign the slides, use a pen capable for cryopreservation or a pen for scratching on glass.

References
Ames, B. N., McCann, J., and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with Salmonella/mammalian microsome mutagenicity test. Mutat. Res. 31, 347-364.

Berry, M. N., and Friend, D. S. (1969). High-yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43, 506-520.

Czygan, P., Greim, H., Garro, J., Hutterer, E, Schaffner, F., Popper, H., Rosenthal, P., and Cooper, D. Y. (1973). Microsomal metabolism of dimethylnitrosamine and the cytochrom P-450 dependency of its activition to a mutagen. Cancer Res. 33, 2983-2986.

Fenech, M. (2000). The in vitro micronucleus technique. Mutat. Res. 455, 81-95.

Fenech, M., and Morley, A. A. (1985). Measurement of micronuclei in lymphocytes. Mutat. Res. 147, 29-36.

Lechner, J. F., LaVeck, M. A., Gerwin, B. I., and Matis, E. A. (1989). Differential responses to growth factors by normal human mesothelial cultures from individual donors. J. Cell Physiol. 139(2), 295-300.

Lovell, D. E, Albanese, R., Clare, G., Richold, M., Savage, J. R. K., Anderson, D., Amphlett, G. E., Ferguson, R., and Papworth, D. G. (1989). Statistical analysis of in vivo cytogenetic assays. In "Statistical Evaluation of Mutagenicity Test Data" (D. J. Kirkland, ed.). Cambridge Univ. Press, Cambridge.

M611er, E, Stock, T., Bauer, S., and Wolff, I. (2002). Genotoxicological characterisation of complex mixtures: Genotoxic effects of a complex mixture of perhalogenated hydrocarbons. Mutat. Res. 515, 99-109.

Natarajan, A. T., Tates, A. D., van Buul, P. P. W., Meijers, M., and de Vogel, N. (1976). Cytogenetic effects of mutagens / carcinogens after activation in a microsomal system in vitro. I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamin (DMN) in CHO cells in the presence of rat-liver microsomes. Murat. Res. 37, 83-90.

Olive, P. L., Banath, J. P., and Durand, R. E. (1990). Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells using the "comet" assay. Radiat. Res. 122, 86-94.

Seglen, P. O. (1976). Preparation of isolated rat liver cells. Methods Cell Biol. XIII, 29-83.

Sgura, A., Antoccia, A., Ramirez, M. J., Macros, R., Tanzerella, C., and Degrassi, F. (1997). Micronuclei, centromere-positive micronuclei and chromosome nondisjunction in cytogenesis blocked human lymphocytes following mitomycin C or vincristine treatment. Mutat. Res. 392, 97-107.

Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, A. L. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184-191.

Stock, T. (2002). Culture of normal human bronchial epithelial cells and its test for toxicological application. Martin Luther University Halle-Wittenberg, medical thesis. (http://sundoc. bibliothek.uni-halle.de/diss-online/02/02H153/index.htm)

Surrall6s, J., and Natarajan, A. T. (1997). Human lymphocytes micronucleus assay in Europe. An international survey. Mutat. Res. 392, 165-174.

Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.-C., and Sasaki, Y. F. (2000). Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206-221.