The flow cytometric MNT provided by Gentronix uses the MicroFlow® dual staining approach for assessment of the induction of micronuclei in TK6 cells. In this method,chromatin from cells undergoing apoptosis/necrosis is initially stained with a light activated, fluorescent, nucleic acid-binding, cell permeability stain.
Following the staining of apoptotic/necrotic cells, cells are lysed to liberate nuclei and micronuclei and the total DNA content is labelled with a second fluorescent nuclei acid stain. Each test chemical dose is then scored for the induction of micronuclei using a flow cytometer.
Sixteen 2-fold dilutions of each test chemical, together with positive and negative controls are set out in a 24-well microplate. TK6 cells in the exponential growth phase are added to the microplate and exposed to test chemicals for between 1.5 and 2 population doublings (typically 26 – 30 hours). After this period, cells are processed for the assessment of micronuclei induction using the MicroFlow® methodology and assessed using flow cytometry.
For assays incorporating metabolic activation through the use of S9, TK6 cells are exposed to test chemical for a reduced period of 3 hours. Following this ‘exposure period’, S9 is removed through a series of cell wash steps and then incubated until cells have reached between 1.5 and 2 population doublings and processed for flow cytometry assessment.
Flow cytometric data collection
A gating methodology is applied that removes events that are ‘dual-stained’ and determined to be from apoptotic/necrotic cells or from events that have abnormal fluorescence and light scatter properties. Following this gating approach, nucleated events are positioned such that the fluorescence intensity is 10 to 100 times higher than micronuclei events (see bivariate plot). Ten thousand nucleated events are collected per dose and a micronucleus frequency is calculated.