I am trying to study the migration of primary cells from explants and hence take long time-lapse movies. Every time I experiment, I need to take an explant and put it in the culture dish and wait for 6 hours before I start imaging the cells. I add the drug 5 minutes before I image. I cannot image the vehicle control and treatment from the same explant, hence for each of the conditions, I need to take an explant every time. The movies are ~2 hours long, and the cells migrate to a very significant distance in this time (about a hundred times the cell length).
In a day, I take two explants. However, the chances of cells coming out from the explant are not 100%, and sometimes, I do not get cells. Thus sometimes, there may occur a gap of a day or two between one control and one treatment. Each two-hour movie represents one data point in my experiments. I require 15 such data points for each condition.
Is this an appropriate experimental design for studying cell migration? In what way can I improve the design?
Hi Alekhya Hati ,
I think you should have access to a microscope with an automated stage. Then you should set up two cell culture vessels from your explants (if you are harvesting only one cut it in half - so the samples are even paired in your statistical analysis). If your cell culture vessel holder at the microscope is not able to hold two vessels at the same time try something like a multiwell plate or µ-Dish 35 mm Quad from ibidi. Then I would recommend to mark multiple positions in your acquisition software to acquire alternating time lapse stacks from all of your positions. In my experience (depending on the speed of your cells and the magnification) a 3-5 min/frame should work.
If your acquisition software does not support this type of experiment (because you have not bought the right license for Zeiss Axiovision it would "Mark and Find" beside the "time lapse" license) you should give the micro manager a try specially if you have an older microscope set up.
Best wishes
Soenke
Hi Sönke Weinert
I do not have access to an automated stage-containing microscope.
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