After co-culturing adherent cancer cells and suspension cells (Jurkat), I want to harvest the Jurkat cells for flow cytometry (FACS). However, the two types of cells are strongly attached to each other. I would like to detach the Jurkat cells without using trypsin. It is acceptable if the cancer cells are also detached during the process. My main goal is to measure CD69 expression on Jurkat cells.
Here are the two methods I’m considering:
Which method would allow me to harvest Jurkat cells without affecting CD69 expression? If you have experience performing FACS after co-culturing adherent cells and immune cells, I would greatly appreciate it if you could share your insights or tips.
Thank you.
Hello Hyunmin Jo,
When Jurkat cells are cultured alongside adherent cancer cells, Jurkat cells can be easily separated from the adherent cells by simply removing the culture medium and washing the adherent cells away, leaving the Jurkat cells in suspension.
While Jurkat cells don't adhere strongly, they can form temporary or weak adhesive contacts with surfaces. In such cases, these cells can be lifted from the dish simply by spraying with phosphate buffered saline and tapping the dish.
Because Jurkat cells are not adherent, there is no need to use trypsin. Trypsin's proteolytic activity can lead to the degradation or modification of cell surface receptors which is likely to affect the experimental results.
The two methods considered by you are appropriate. But I would suggest that you use EDTA instead of EGTA. While EGTA is a calcium chelator with a higher affinity for calcium than EDTA, its primary use is in cell biology research to study calcium signaling pathways, rather than it's use in cell detachment.
Finally, to study surface markers in co-cultures, as far as possible, aim for a pure population of cells.
Good Luck!
Why not simply try using CD3⁺CD69⁺(if the antibody is available) via flow cytometry? You never know how EGTA might affect your cells, and it could raise concerns. I suggest avoiding unnecessary complications that could lead reviewers to question the certainty of your results — such as potential cell contamination or the impact of EGTA as a contributing factor.
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