I'm hoping to do a clonal expansion of Jurkat, expressing my construct which is currently transduced virally. From the FACS data I currently have roughly 30-40% expression in my culture but I need it to be close to 100% as the effect I'm looking for I believe to get diluted by the reduced presence of my molecule of interest in the total culture. I can do cell sorting but usually what happens is that the 5% cell population that do not express the construct eventually take over after a few passages.

I'm planning to do a 96 well plate format single cell seeding and expansion of Jurkats but it has been done before by another member of our lab and the person suggests that single cell Jurkats (unlike CHO cells) do not survive a single cell state although they are suspension cells and do not necessarily require contact to survive although they clump. Hence I'm wondering if anyone has any protocols they could suggest such as contents of the media (e.g. conditioned media) or any other recommendations for Jurkat single cell clonal expansion.

P.S. I am fully aware that there might not be a single right answer to this and the answer requires a degree of experience/opinion on the working method but I would fully appreciate any helpful suggestions/responses.

Many thanks

  • $\begingroup$ Is there a chance to use a selection marker like G418 to kill all the cells which do not express your construct? $\endgroup$ – Chris Aug 20 '15 at 10:15
  • $\begingroup$ I will look to see if the vector has this otherwise a fresh co-transduction or a new vector containing antibiotic resistance selection marker containing something like G418 might be required. I think people are unsure as to exactly what backbone the construct is in so that could be tricky and require month of cloning and sub-cloning. Are Jurkats tend to be difficult with clonal expansion? $\endgroup$ – Behzad Rowshanravan Aug 20 '15 at 10:21
  • $\begingroup$ Jurkats shouldn't be difficult to clonally expand. If you're having problems, get a fresh culture from ATCC. You really also should know what kind of vector you're using - if you don't know what your reagents are, there is no way to independently verify your results. Therefore, they are invalid. $\endgroup$ – MattDMo Aug 20 '15 at 14:47

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