I am trying to perform a cell aggregation assay with CHO cells transfected with cell adhesion molecules. However, the control cells show the same high level of aggregation. I saw a number of publications in which naive CHO cells did not aggregate. Is there a trick to this?
You could try adding substances such as dextran sulfate to your media which has been reported to prevent the aggregation.
Are you growing your cells as adhesion or suspension culture?
Thank you, David. I'll try that. I am growing them as an adhesion culture.
Since some CHO cell strains form tight colonies during their growth, and don't move around much on the plate, all of the cells may appear to be "aggregating" with other cells in their colony. If the plate is agitated regularly, the colonies tend to break up and be more diffuse, which may help distinguish the cells truly aggregating with each other from those just sitting closely with their intra-colony sister cells.
John,
Although the cells are grown as adhesion culture, for the aggregation assay, they are detached, dispersed and incubated as a suspension on a rotary shaker. Based on the literature, they shouldn't aggregate, unless they are transfected with a cell adhesion molecule. In my hands they do, and a lot.
Got it, it sounds like you are doing it correctly, and under those conditions they should not be aggregating as long as your initial detachment and dispersal steps are thorough. Perhaps some extra time with the trypsin/EDTA on the detachment step might break them up a little bit more thoroughly. Good luck, Jana.
Further, sometimes DNA released from dead cells can lead to a clumping of cells. DNase I treatment has helped in some cases.
Other than that I agree with Olivia's and John's suggestions.
Good luck with the assay!
- Badges
- Science method