Quantifying Epigenetic Changes Induced by Chemical Exposure Using the epi-TK Assay

Materials and reagents

Biological materials

1. Human lymphoblastoid mTK6 cell line (TK6-derived cell line originally established in a previous study [6]; available from the authors upon request), stored at -80 °C for short-term storage or in liquid nitrogen for long-term storage. Alternatively, equivalent cell lines can be established from the parental TK6 cell line or other cell types by isolating TFT-resistant clones generated via transient expression of dCas9-DNMT3A with guide RNAs targeting the TK gene promoter [6].

Reagents

1. RPMI-1640 medium (Nacalai Tesque, catalog number: 30264-56); store at 4 °C

2. Fetal bovine serum (FBS) (Nichirei Biosciences, catalog number: 174012-500ML); aliquot and store at -20 °C

3. 100 mM sodium pyruvate solution (Nacalai Tesque, catalog number: 06977-34); store at 4 °C

4. Penicillin-streptomycin solution (Nacalai Tesque, catalog number: 09367-34); store at 4 °C

5. Trifluorothymidine (TFT) (Sigma-Aldrich, catalog number: T2255-100MG)

6. 2′-Deoxycytidine hydrochloride (Sigma-Aldrich, catalog number: D0776)

7. Hypoxanthine (Sigma-Aldrich, catalog number: H9377)

8. Aminopterin Hybri-Max^TM (Sigma-Aldrich, catalog number: A5159)

9. Thymidine (Sigma-Aldrich, catalog number: T9250)

10. GSK-3484862 (CHEMIETEK, catalog number: CT-GSKMI)

11. Dimethyl sulfoxide (DMSO) (Nacalai Tesque, catalog number: 13408-64)

12. Hydrochloric acid (HCl) (Nacalai Tesque, catalog number: 18321-05)

Solutions

1. RPMI-1640 complete medium (see Recipes)

2. 1,000× TFT (see Recipes)

3. 1,000× 2′-deoxycytidine (see Recipes)

4. 1,000× hypoxanthine (see Recipes)

5. 1,000× thymidine (see Recipes)

6. 100× CHAT (2′-deoxyCytidine, hypoxanthine, aminopterin, and thymidine) (see Recipes)

Recipes

1. RPMI-1640 complete medium

Store at 4 °C.

2. 1,000× TFT

Sterilize by filtration through a Minisart 0.2-μm syringe filter and store at -20 °C.

3. 1,000× 2′-deoxycytidine

Sterilize by filtration through a Minisart 0.2-μm syringe filter and store at -20 °C.

4. 1,000× hypoxanthine

Sterilize by filtration through a Minisart 0.2-μm syringe filter and store at -20 °C.

5. 1,000× thymidine

Sterilize by filtration through a Minisart 0.2-μm syringe filter and store at -20 °C.

6. 100× CHAT

Sterilize by filtration through a Minisart 0.2-μm syringe filter and store at -20 °C.

Laboratory supplies

1. 10-cm culture dish (AS ONE, catalog number: 1-7484-01)

2. 15-cm culture dish (SPL Life Science, catalog number: 20150)

3. 15- and 50-mL conical tubes (Greiner Bio-One, catalog numbers: 188271-N and 227261)

4. 1.5-mL microcentrifuge tube [Scientific Specialities Inc (SSI bio), catalog number: 1210-10]

5. Reagent reservoir (Greiner Bio-One, catalog number: J908305)

6. Serological pipettes (5, 10, 25, and 50 mL) (Greiner Bio-One, catalog numbers: 606180, 607180, 760180, and 768180)

7. 96-well flat-bottom plate (VIOLAMO, catalog number: 1-1610-06)

8. Minisart 0.2-μm syringe filter (Sartorius, catalog number: S7597FXOSK)

Equipment

1. Pipetman P10, P20, P200, and P1000 (Gilson, catalog numbers: F144055M, F144056M, F144058M, and F144059M)

2. P200L multichannel micropipette (Gilson, catalog number: FA10011)

3. Pipet-Aid XP (Drummond, catalog number: 4-040-101-J)

4. Inverted microscope (Carl Zeiss, catalog number: Primovert)

5. Stereomicroscope (Leica, catalog number: M205 C)

6. Biological safety cabinet (Panasonic Healthcare, catalog number: MHE-S1301A2)

7. CO₂ incubator (Panasonic Healthcare, catalog number: MCO-19AIC)

8. Multitube refrigerated centrifuge (TOMY SEIKO, catalog number: AX-511)

9. Beckman Coulter Z2 particle counter (Beckman Coulter, catalog number: 6605700)

10. Counting chamber (Erma, catalog number: 10-2170-05)

Procedure

A. Preparation of cell cultures

Note: All steps must be performed in a biological safety cabinet.

1. Seed mTK6 cells at a density of 30,000 cells/mL in a 15-cm culture dish containing RPMI-1640 complete medium.

2. Add 1/1,000 volume of 1,000× TFT solution to the culture medium and incubate the cells for 3 days at 37 °C in a humidified CO₂ incubator with 5% CO₂.

Note: TFT is added to the culture medium during initial culture to suppress the gradual accumulation of spontaneous TK revertants, thereby maintaining a stable baseline revertant frequency over serial passages.

3. Examine cell morphology and assess contamination using an inverted microscope. Transfer the cell suspension to a 50-mL conical tube and determine the cell number using a Z2 particle counter or a counting chamber.

4. Calculate the appropriate dilution factor to adjust the cell density to 200,000–300,000 cells/mL. Seed the cells into a new 15-cm culture dish containing fresh RPMI-1640 complete medium and add 1,000× TFT solution at 1/1,000 of the final culture volume. Incubate the cells for an additional 24 h under the same conditions.

B. Cell preparation for chemical exposure

Note: All steps must be performed in a biological safety cabinet.

1. Transfer the entire culture volume to a 50-mL conical tube and determine cell density using a Z2 particle counter or counting chamber. At this stage, a cell density of approximately 800,000–1,000,000 cells/mL is recommended.

2. Based on the measured cell density, transfer a volume of the cell suspension corresponding to (number of treated groups + 1) × 2,500,000 cells into a new 50-mL conical tube.

3. Centrifuge the cells at 500× g for 5 min at 4 °C using a multitube cooling centrifuge. Carefully discard the supernatant containing RPMI-1640 complete medium with TFT and gently loosen the cell pellet by tapping.

4. Add fresh RPMI-1640 complete medium without TFT at a volume of (number of treated groups + 1) × 5 mL and resuspend the cells thoroughly.

5. Measure the cell density using a Z2 particle counter or a counting chamber and adjust the concentration to 500,000 cells/mL ± 5% (i.e., 475,000–525,000 cells/mL).

6. Aliquot 5.0 mL of the cell suspension into 15-mL conical tubes and add 4.9 mL of fresh RPMI-1640 complete medium to each tube. Add 100 μL of the test reagent (e.g., GSK3484862; final concentrations: 0.5, 2, or 5 μM) or 100 μL of DMSO as a vehicle control. Test reagent concentrations should be adjusted so that the cloning efficiency (CE, described below) of treated cells remains within approximately 20%–100% of the vehicle control, because cell proliferation during the treatment period is required to reveal epigenetic alterations. For compounds with unknown cytotoxicity, it is recommended to first evaluate a concentration range starting from 10 mM (or 2 mg/mL for compounds with molecular weight >200), typically using serial 10-fold dilutions, to identify concentrations suitable for the assay.

7. Transfer the contents of each 15-mL tube to 10-cm culture dishes by decanting and incubate the cells for 24 h at 37 °C in a humidified CO₂ incubator with 5% CO₂.

C. Cell washing, cell density adjustment, and plating

Note: All steps must be performed in a biological safety cabinet.

1. Transfer the entire cell suspension from each 10-cm dish into a 15-mL conical tube and centrifuge at 500× g for 5 min at 4 °C.

2. Discard the supernatant by decanting, gently loosen the cell pellet by tapping, add 5 mL of RPMI-1640 complete medium, mix by gentle inversion, and centrifuge at 500× g for 5 min at 4 °C.

3. Repeat step C2 once.

4. Discard the supernatant, gently loosen the cell pellet, resuspend the cells in RPMI-1640 complete medium, and determine the cell density using a Z2 particle counter or a counting chamber.

5. Based on the measured cell density, adjust each treatment group to a final concentration of approximately 200,000 cells/mL in a total volume of 10 mL using fresh RPMI-1640 complete medium.

6. Prepare serial dilutions of the cell suspension for plating on revertant frequency (RF) plates (two plates per treatment group) and cloning efficiency (CE) plates (one plate per treatment group), as shown in Tables 1 and 2 for the vehicle control and chemical treatment groups, respectively.

Table 1. Dilution scheme for vehicle control

Note: Cells from the vehicle control group are serially diluted from an initial concentration of 2.0 × 10⁵ cells/mL to prepare cell suspensions for revertant frequency (RF) plating at 5,000 cells/mL and cloning efficiency (CE) assays at 8 cells/mL.

Table 2. Dilution scheme for the GSK3484862-treated groups

Note: For the GSK3484862-treated groups, cells are serially diluted from an initial concentration of 2.0 × 10⁵ cells/mL through an intermediate dilution step at 5,000 cells/mL to prepare cell suspensions for revertant frequency (RF) plating at 100 cells/mL and cloning efficiency (CE) plating at 8 cells/mL. For compounds with unknown epigenetic activity, it is recommended to preliminarily examine RF plating densities over a range of 100–100,000 cells/mL using serial 10-fold dilutions to identify conditions that yield interpretable colony numbers.

7. After completing all dilution steps, add 100× CHAT solution at 1/100 of the final culture volume prepared for RF plating. In the present protocol, it corresponds to 500 μL for the vehicle control tube and 485 μL for the GSK3484862-treated tubes, based on their respective final suspension volumes shown in Tables 1 and 2.

8. Transfer the RF or CE cell suspensions to reagent reservoirs and seed 200 μL per well into 96-well flat-bottom plates using a P200L multichannel micropipette.

9. Incubate the plates for 3 weeks at 37 °C in a humidified CO₂ incubator with 5% CO₂.

D. Colony counting and data analysis

1. Count the number of colonies on the CE and RF plates separately. First, place the plates on a white background and mark the wells in which the culture medium has changed color from red to yellow, indicating colony formation (Figure 1A).

2. Next, inspect each well directly under a stereomicroscope to confirm whether colonies are present in wells that were not marked in step D1. Colonies are observed as discrete cell aggregates within the wells, as shown in Figure 1B. Color change of the medium should not be used as the sole criterion for colony scoring and should be confirmed by microscopic observation to ensure accurate results.

3. Record the number of colonies for the CE and RF plates according to Tables 3 and 4.

4. Calculate CE using equation (1), based on the Poisson distribution [8], where EW represents the number of wells without colonies, TW represents the total number of wells, and N is the average number of cells per well (N = 1.6) in the CE plates (Table 3). Relative survival (%) is determined by comparing the CE values of chemically treated cells with those of the vehicle control.

CE = -ln (EW/TW)/N            (1)

5. Calculate revertant frequency (RF) using equation (2), also based on the Poisson distribution, where N corresponds to the number of cells per well in the RF plates.

RF = [-ln (EW/TW)/N]/CE_treated    (2)

Table 3. Cloning efficiency and relative survival of mTK6 cells following treatment with GSK-3484862

Table 4. TK revertant frequency of mTK6 cells following treatment with GSK-3484862

6. Plot the results by displaying chemical concentration on the x-axis and relative survival and TK revertant frequency on the y-axis, as shown in Figure 2A, B.

7. Repeat all experiments independently at least three times and statistically analyze the data using Dunnett’s test by comparing chemical-treated groups with the vehicle control.

Figure 1. Representative images of cloning efficiency and revertant frequency plates generated in the epi-TK assay. (A) Representative images of cloning efficiency (CE) (left) and revertant frequency (RF) plates (right) prepared following chemical treatment. CE plates were used to determine cell survival, whereas RF plates were used to quantify revertant frequency. (B) Representative microscopic images of negative wells (no colony formation) and positive wells (visible colony formation). Wells were scored based on the presence or absence of colonies and used for Poisson-based calculation of CE and RF values.

Figure 2. Relative cell survival and thymidine kinase (TK) revertant frequency measured using the epi-TK assay following GSK3484862 treatment. Cytotoxic and epigenetic effects were measured as relative cell survival (A) and TK revertant frequency (B), respectively, following treatment with GSK-3484862. The data are representative of a single independent experiment.

Validation of protocol

This protocol has been applied and validated in the following research article:

1. Yamada et al. [6]. Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene. Scientific reports (Figure 2).

General notes and troubleshooting

General notes

1. In this protocol, CHAT medium is used for selection. Although hypoxanthine–aminopterin–thymidine medium supports selection of TK revertants, CHAT provides more stable colony formation and improved reproducibility by supplementing pyrimidine metabolism with cytidine.

2. Spontaneous revertant frequency may vary slightly depending on the solvent used; therefore, a vehicle control should be included for each test chemical.

3. For chemical-treated groups, the RF plating density should be optimized based on the anticipated epigenetic effects of the test compound. Compounds that promote DNA methylation, inhibit demethylation, or enhance heterochromatin formation may require higher RF plating densities than the vehicle control. In contrast, compounds that inhibit DNA methylation, promote demethylation, or enhance euchromatin formation may require lower RF cell densities than the vehicle control. The plating density used in this protocol was selected to obtain approximately 10–70 colonies per 96-well plate under baseline conditions, which provides reliable revertant frequency calculation based on the Poisson distribution. This density also allows stable detection of both increased revertant frequency induced by DNA demethylating agents and reduced colony formation caused by compounds affecting chromatin regulation.

4. Chemical treatment conditions that result in excessive cytotoxicity impair cell proliferation and may compromise RF colony formation. Because cell proliferation during the treatment period is required to reveal epigenetic alterations, test compound concentrations should be selected to maintain a relative CE of at least 20% compared with the vehicle control. As a preliminary optimization step, cells may be washed to remove the compound after treatment and cultured for an additional 24 h, after which cell proliferation can be assessed.

5. Horse serum (HS) can be used as an alternative to FBS. The RF colony formation typically proceeds more slowly in HS- supplemented cultures than in FBS-supplemented cultures; however, an incubation period of up to 3 weeks is sufficient to allow visible colony formation under either condition.

6. When metabolic activation is required (e.g., through the use of an S9 mix), the chemical treatment duration should be reduced from 24 to 4 h. Following treatment, cells should be washed to remove both the test compound and the S9 mix, followed by a 24-h recovery period before proceeding with CE and RF plating.

7. Our previous findings indicate that the methylated TK locus is enriched in histone acetylation marks and therefore appears unresponsive to histone deacetylase inhibitors that generally induce chromatin relaxation [6]. In contrast, compounds causing global reduction of histone acetylation produce measurable changes in TK revertant frequency, supporting the utility of this assay for detecting such epigenetic effects.

Troubleshooting

Problem 1: Fungal contamination is observed in some wells of the CE or RF plates.

Possible cause: Due to the extended incubation period (up to 3 weeks), fungal contamination may occur depending on CO₂ incubator conditions.

Solution: If fungal contamination is detected, exclude the affected wells from analysis and calculate CE and RF values using only uncontaminated wells.

Problem 2: Colonies are observed in all wells of the RF plates.

Possible cause: CHAT selection pressure is insufficient to restrict colony formation to TK revertant cells. Alternatively, the cell density for plating is inappropriate for detecting the epigenetic effects of the test compound.

Solution: Aminopterin is available from multiple suppliers and in various grades; the performance of alternative products has not been validated. Prepare CHAT strictly according to the protocol described in the Recipes section. In addition, optimize the RF plating density within a range of 100–100,000 cells/mL, depending on the expected epigenetic mode of action of the test compound.

Acknowledgments

The authors thank Dr. Kiyoe Ura and Mr. Koh Iwasaki (Chiba University) for their valuable comments, discussions, and technical assistance. This work was supported by the Consumer Affairs Agency, Government of Japan (Grant Number 24KA1008), the Ministry of Health, Labour and Welfare of Japan (Grant Number 21KA1001), and JSPS KAKENHI (Grant Number 22H03748).

Conceptualization, S.K., H.Y., M.O., M.Y., and A.S.; Investigation, S.K., H.Y., M.O., K.S., M.Y., and A.S.; Original Draft Preparation, S.K. and A.S.; Writing—Review & Editing, S.K., H.Y., M.O., K.S., M.Y., and A.S.; Funding Acquisition, K.S., M.Y., and A.S.; Supervision, M.Y. and A.S.

This protocol was adapted from procedures published in Scientific Reports (2025), DOI: 10.1038/s41598-025-92121-6.

Competing interests

The authors declare that they have no competing interests.

References

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