Troubleshooting

In Vitro MicroFlow Kits

Why are the negative control values for micronuclei, or EMA-positive nuclei, higher than expected?

This is likely due to cell culture conditions, which can have a significant impact on micronuclei and EMA-positive values.

Possible Cause:
The actual doubling time of your cell culture is different than expected.

Suggestion:
Establish a growth curve to confirm the doubling time of your culture both after thaw and during routine cell passage. Wait at least one week after thaw before treatment to allow the cells to equilibrate.

Use your calculated doubling time to determine the correct time to harvest cells (see next question).


Possible Cause:
The media, serum, antibiotics, or other growth factors are not appropriate for your cell line.

Suggestion:
Ensure that you are using cell culture media and supplements that are appropriate for your cell line.


Possible Cause:
The growth environment is not optimal.

Suggestion:
Maintain an incubation temperature of 37 °C, CO2 levels at 5 %, and humidity near 88 %.


Possible Cause:
The cells are overgrown, which can increase micronuclei and EMA-positive values.

Suggestion:
Ensure that cell cultures do not exceed the following:

Suspension cells: 1.0 x 106 cells/ml
Attachment cells: 80 % confluence

Why are my positive controls not showing a dose related response?

Possible Cause:
Cells may have been harvested too early, resulting in too few cell divisions for micronuclei to be expressed.

Suggestion:
OECD indicates cells should be treated for 1.5 to 2.0 doublings before harvest to allow for proper expression of micronuclei. Use the doubling time you calculated for your cell culture to determine the appropriate harvest time (see previous question).

How do I determine Percent Relative Survival (% RS) using Nuclei:Bead ratios?

You can calculate % RS using the following formula:

% RS = (Treatment Nuclei:Bead Ratio X 100) / Negative Control Nuclei:Bead Ratio

 

Why is the FITC channel shifting when using the High Throughput System (HTS) on a BD flow cytometer?

Possible Cause:
It is our experience that shifts in the FITC channel when using an HTS on a BD flow cytometer are most likely caused by the type of sheath used.
 
Suggestion:
To solve this problem, switch to water or a blood bank saline solution (like NERL from Fisher Scientific) for your sheath.

Don't see your question here?

Feel free to send us new questions or example plots to examine. We are always here to help!

Contact Us