An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The image stream-based micronucleus assay can overcome several limitations of methods such as automated microscopy and conventional flow cytometry, including low throughput and lack of visual confirmation of events. The main advantage of this technique is that all required events to determine genotoxicity and cytotoxicity can be automatically imaged, identified, and scored without the need to create microscope slides. For cell exposure to clastogens and/or aneugens, add one millimeter of the chemical of interest to seven to eight times 10 to the fifth of the experimental cells in nine milliliters of the appropriate cell culture medium in a T25 flask and one milliliter of water to the control cultures.

Incubate the flasks at 37 degrees Celsius and five percent carbon dioxide for three hours before transferring the cells to one 15 milliliter polypropylene tube per flask. Collect the cells by centrifugation and resuspend the pellets in 10 milliliters of fresh culture medium per tube for seeding in new T25 culture flasks. Then add 150 microliters of the stock concentration of cytochalasin B to each flask to achieve a final concentration of three micrograms per milliliter.

Return the flasks to the incubator for a recovery time equal to 1.5 to two doubling times, as recommended by the Organization for Economic Cooperation and Development guidelines. At the end of the recovery period, transfer the cultures into individual 15 milliliter polypropylene tubes for centrifugation and resuspend the pellets in five milliliters of 75 millimolar potassium chloride. Mix gently by inversion three times and incubate the samples at four degrees Celsius for seven minutes.

At the end of the incubation, add two milliliters of four percent formalin to each sample and mix gently with three inversions. Return the samples to four degrees Celsius for 10 minutes before collecting the cells by centrifugation. resuspend the pellets in 100 microliters of fresh four percent formalin for 20 minutes.

At the end of the incubation, wash the fixed cells in five milliliters of wash buffer per tube by centrifugation and resuspend the pellets in 100 microliters of wash buffer per tube. Next, transfer the samples to individual 1.5 milliliter microcentrifuge tubes for counting on a hemocytometer. Add five microliters of 100 micrograms per milliliter Hoechst 33342 per 1.0 x 10 to the six cells per milliliter and 10 microliters of 500 micrograms per milliliter RNase per 100 microliters of sample per tube.

After a 30 minute incubation at 37 degrees Celsius and five percent carbon dioxide, centrifuge the samples in a microcentrifuge and remove all but 30 microliters of supernatant from each tube. Then carefully resuspend the samples, taking care not to induce bubble formation. If bubbles do appear upon resuspension, gently flick the tube to remove them.

To run the samples by multispectral imaging flow cytometry, first turn on the 405 nanometer laser and set the laser power to 10 milliwatts. Disable all of the other lasers, including the side scatter laser, and set the brightfield to channels one and nine. Confirm that the magnification slider is set to 60x, that the high sensitivity mode is selected, and that only channels one, seven, and nine are showing in the image gallery.

Click scatter plot and select the all population and area M01 on the X axis. Select aspect ratio M01 on the Y axis and click square region to draw a region around the single cells. Name this region single cells and right click on the plot to select regions.

Highlight the single cells region and change the X coordinates to 100 and 900 and the Y coordinates to 0.75 and one. Set the acquisition parameters and specify the file name and the destination folder. Then change the number of events to 20, 000, select the DNA positive population, and run the first sample.

To open a data file in IDEAS, click Start Analysis to start the Open File Wizard, and browse to select the desired raw image file. Then click Next and browse for a binucleated cell in the image gallery. To create a mask, click to select the image of interest and open the analysis tab.

Click Masks, New, and Function to select LevelSet. Under Mask, select M07 and Middle Level Mask and set the contour details scale to three. Then click OK two times.

To spot count binucleated cell features, open the Analysis tab and select Features and New. For the Feature Type, select Spot Count. For Mask, select the final binucleated mask, and set the connectiveness to four, then change the name to Spot Count BNC and click OK and close to calculate the feature values.

For a spot count binucleated cell histogram, click histogram and select non-apoptopic as the parent population. For the X axis feature, select Spot Count BNC and click OK and linear region, then draw a region across Bim Two and name this region 2N. To create a micronucleus mask, select a binucleated cell that contains a micronucleus from the image gallery, and open masks in the Analysis tab.

To create a spot identification mask one, click New and Function, and select Spot. Confirm that the Bright radio button is selected, and select M07 under mask. Set the Spot to Cell Background Ratio and the Minimum Radius to two and set the Maximum Radius to six.

Click OK and OK to add the mask to the list on the left, then click New to create another mask. Click the Spot M07 Channel seven Bright two six two mask and click the down arrow to add it to the mask definition and click the And and Not operators. Click the down arrow to add dilated range mask to the mask definition.

To create a polynucleated population mask, click Analysis, Masks, New, and Function. Under Function, select Range. Under Mask, select Watershed Dilate Set M07 Channel07, Middle, three, two, and set the image to display to Channel 07.

Set the minimum and maximum area values to 135 and 5000, respectively, and set the minimum and maximum aspect ratio values to 0.4 and one, respectively. Click OK and in the name field, change the text to read POLY mask, then click OK and Close. To create a custom view, click the Image Gallery Properties button, open the Composites tab, and click New.

Under Name, enter Channel 01 to Channel 07. Click Add Image, and under Image, select Channel 01 and set the percent to 100. Click Add Image again, and under image, select Channel 07 and set the percent to 100.

Click the View tab, click New, and under Name, enter BNC and MN masks. Then click Add Column, and under Image type, select Channel 01 and under Mask, select None. Click Add Column, and under Image Type, select Channel 07, and under Mask, select None.

Click Add Column, click the Composite radio button, then click Okay to complete the custom view. Click the View pulldown menu, and click the BNC and MN masks view. Click the Show hide masks button.

To enumerate key events, open the Reports tab, and click Define Statistics Report, then in the new window, click Add Columns. To add the binucleated cell counts statistic, under Statistics, select Count, and under Selected Population, select the BNC population, then click Add Statistics to add the statistic to the list and save the template. Once all statistics have been added to the list, click Close, then click OK, then save the data analysis template.

To batch process the experimental files, under the Tools menu, click Batch Data Files and click Add Batch in the new window. Click Add Files to select the experiment files to add to the batch and click the Open Folder button under the Select a template or data analysis file option. Browse to the template that has just been saved and open it.

Click OK to close the current window. Then click Submit Batches to start the batch processing of all files. Here, four selected panels for identifying binucleated cells are shown.

These bivariate plots and histograms enable the selection of binucleated cells as well as the identification of those cells that have two nuclei with similar circularity, areas, and intensities and that are well separated from one another. These Brightfield and Hoechst images, as well as the binucleated cell and micronucleus masks, facilitate the identification and enumeration of binucleated cells and micronuclei. Application of the spot count feature uses the polynucleated mask to identify mononuclear, trinuclear, and quadranuclear cells.

The number of tri and quadranuclear cells can then be summed to obtain the final number of polynucleated cells required for the calculation of cytotoxicity. Here, representative genotoxicity and cytotoxicity values are shown for the endogen colchicine, the clastogen mitomycin C, and a negative control, mannitol, to demonstrate the validity of the protocol. Labeling the cells with an appropriate concentration of DNA stain is critical to ensure high quality image capture, permitting all key events to be easily identified and scored.

These techniques are also applicable to the micronucleus assay for radiation biodosimetry, which allows dose estimation in individuals who may have been exposed to radiation.