This site uses cookies. By continuing to browse the site you are agreeing to our use of cookies. Find out more here.
ADME PK

In vitro Micronucleus Test (MNT; Flow Cytometry TK6 Cells)

Investigate genotoxicity using a flow cytometry based micronucleus test in TK6 cells.

Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Assess genotoxicity using TK6 cells and flow cytometry

  • Two key classes of genotoxic agents are clastogens and aneugens. Clastogens directly damage DNA resulting in double strand DNA breaks. Aneugens produce numerical chromosome aberrations (aneuploidy), the result of “lagging” chromosomes.
  • The actions of both anuegens and clastogens result in the formation of micronuclei within daughter cells following cell division.
  • Flow cytometry allows multiparametric analysis of compound effects including cytostasis, cellular death and cell cycle arrest in addition to micronucleus formation.
  • Flow cytometry quantifies marker intensity (Hoechst, CFDA-SE, EMA) and object size to detect micronuclei.
  • Exclusion of necrotic and late apoptotic cells from analysis reduces false positives for genotoxicity. Monitoring of cytostasis and exclusion of cytostatic cells from micronucleus analysis also helps to reduce false negative results.
  • The human lymphoblast line TK6 expresses the wild type p53 protein, shown to be important in accurate genotoxicity prediction1.
The in vitro MNT allows the detection of both clastogen and aneugens and it can simultaneously detect mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction.

2Corvi R, Albertini S, Hartung T, Hoffmann S, Maurici D, Pfuhler S, van Benthem J and Vanparys P (2008) Mutagensis 23(4); 271-283

Protocol

In vitro micronucleus test protocol using TK6 cells and flow cytometry

Test System Flow cytometry using Intellicyte (iQue Screener Plus)
Cell Line TK6 cells (other cells may be available on request)
Metabolizing System With or without S9 fraction
Time Points 3 hours (+S9) and 24 hours (-S9)
Quality Controls Assay appropriate controls
Test Article Requirements 5-10 mg solid or equivalent solution depending upon top test concentration required (200x to maintain 0.5% vehicle).
Endpoints Micronucleus formation, cell death, cytostasis, cell cycle analysis (G1, S, G2/M phase)

Data

Data from Cyprotex's in vitro micronucleus test using TK6 cells and flow cytometry

 
determining-micronuclei
Figure 1
Representative data for micronucleus formation and cell cycle analysis. Cells were treated with vehicle or paclitaxel at the stated concentrations for 24 hours prior to analysis. Micronuclei are detected as shown in figure 1A, with Hoechst staining (VL1) being utilised to identify the nuclei and micronuclei. The nuclei are then categorised as G1, S or G2/M phase (figure 1B).
  -S9+S9Classification
CompoundCategoryMEC  (µM)Max
response (% Control)
MEC
(µM)
Max
response (% Control)
-S9+S9
Methyl methanesulfonate Genotoxic 14.1 159 9.58 61.8 Positive Positive
Etoposide Genotoxic 0.51 102 1.13 73.8 Positive Positive
Methotrexate Genotoxic <0.08 46.1 0.344 15.3 Positive Negative
4-Nitroquinoline N-oxide Genotoxic 0.0094 232 244 22.2 Positive Negative
Mitomycin C Genotoxic <0.04 141 <0.04 266 Positive Positive
Chlorambucil Gentoxic <0.2 123 <0.2 118 Positive Positive
Cyclophosphamide Genotoxic NR NR 2.93 261 Negative* Positive
araC Genotoxic 0.00329 55.9 0.138 23.3 Positive Negative
7-12-Dimethylbenz[a]anthracene Genotoxic 0.24 55.3 0.869 87.3 Positive Positive
Benzo[a]pyrene Genotoxic <1.56 61.3 <1.57 35.7 Positive Positive
Nocodazole Genotoxic <0.04 228 41.4 192 Positive Positive
Vinblastine Genotoxic <0.004 245 <0.004 18.5 Positive Negative
Colchicine Genotoxic <0.004 203 0.0292 227 Positive Positive
Paclitaxel Genotoxic <0.004 65.3 <0.004 291 Positive Positive
Diethylstilbestrol Genotoxic 0.962 195 31.7 2.88 Positive Negative
Griseofulvin Genotoxic 2.44 66.5 NR NR Positive Negative
Dexamethasone Non-genotoxic 27.2 1.45 NR NR Negative Negative
Chlorpromazine Non-genotoxic NR NR NR NR Negative Negative
CCCP Non-genotoxic 1 3.68 34.3 3.52 Negative Negative
Cyclosporin A Non-genotoxic NR NR NR NR Negative Negative
Imatinib Non-gentoxic NR NR 47.2 8.21 Negative Negative
Staurosporine Non-genotoxic 0.0165 9.23 1.22 33.2 Negative Negative
Alendronate Non-genotoxic 35.8 3.07 NR NR Negative Negative
Amoxicillin Non-genotoxic NR NR NR NR Negative Negative
Metformin Non-genotoxic NR NR NR NR Negative Negative
Nalidixic acid Non-genotoxic NR NR NR NR Negative Negative
Pyrene Non-genotoxic NR NR 19 4.53 Negative Negative
Table 1
Summary of data for genotoxic and non-genotoxic reference compounds in the in vitro micronucleus test using TK6 cells and flow cytometry.

TK6 cells were incubated with a panel of compounds in the presence or absence of S9 fraction for 3 or 24 hours respectively prior to analysis. At concentrations where over 30% cellular death or cytostasis was observed any present micronuclei was excluded. Compounds were categorised as genotoxic if they induce a 35 fold increase in micronucleus formation compared to vehicle only controls. Green boxes show correctly predicted compounds.  The positive maximum (Max) response threshold was set at 35 fold increase compared to vehicle only controls. Orange boxes are above this threshold, blue boxes are below the threshold. 

* Cyclophosphamide is metabolically activated.
NR = No Response.

References

1 Kumari R et al., (2014) p53 regulation upon genotoxic stress: intricacies and complexities. Mol Cell Oncol 1(3); DOI: 10.4161/23723548.2014.969653 
2 Corvi R et al. (2008) ECVAM retrospective validation of in vitro micronucleus test. Mutagenesis 23(4); 271-283

Request a Quote
Speak to a Scientist
Product sheet ADME-Tox Guides Pricing & Discounts

Contact us to discuss your ADME Tox issues or request a quote

Telephone:
North America (East Coast): 888-297-7683
Europe: +44 1625 505100

 

or fill out the form below:

Please give details of the assays you are interested in. Where appropriate please specify one or more species (human, rat, mouse etc.), isoforms (CYP1A1,CYP1B1, etc) or other relevant details.

Close