I transduced my cells with 2ml of the viral supernatant and 4ug/ml polybrene. After transduction, 16 hours, I replaced the media with normal media. I started antibiotic selection with 6ug/ml blasticidin 48 hours after transduction. During selection there was a lot of cell death, although compared to my positive control some cells remained in my T75s. I selected for just one week/ when all the cells in my positive control flask were dead. However these cells are in these little cell islands, and there are very few cells. They appear to be slow growing and I am not confident that my flask will become confluent. Can someone tell me what I could have done wrong?
I think you are okay, as some cells survived and are now growing in satellite "colonies". However, I would harvest and replate the cells into a smaller vessel (T25 or 35mm dish) to encourage paracrine signaling amongst them and increase survival. They will likely take time to grow back out to a usable number. Once in a smaller vessel, I would also recommend keeping 20-25% of the conditioned media from the cells and apply 75% fresh to maintain signaling on the happy cells.
Just to keep in mind - the titer of virus determines the multiplicity of infection - the # of particles per cell that are transduced. If this number is too high then it might stress the cells and the cells will die. If this number is too low then there is low efficiency of transduction and only a few cells will survive to then grow out.
Sometimes spinning the plate/flask with the virus can help increase transduction - there will be literature on speeds and times. This might be useful in the future.
Thanks Julie.
This is my second time repeating this transduction. I previously harvested and replaced into a new dish, cells from this did not adhere to the new culture dish. Do you have any idea why this might be?
the cells were likely stressed. be as gentle as possible - if you can use PBS + EDTA to release rather than trypsin I would try that (called enzyme-free cell dissociation buffer or versene or you can look up a recipe and make it, works on cells that are not firmly adhered). pipet very gently.
you could try inoculating cells in a well-plate like 24-well or 96-well, then growing out. or you can put colony isolation rings over the small groups of cells but that would work on a plate, not a flask.
also it depends on the cell type and the dish, perhaps adding an additional coating might help them adhere - the type you use would depend on what is accepted to use for the cell type. poly-d-lysine coating is usually accepted for most cells. otherwise collagen or fibrinogen could work. however, do NOT do this if it is not well-published for your cell type as it would be a new protocol and reviewers will come after you when a paper is submitted.
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