I've been trying to work with the Invivogen mTLR3-293Ts that stably express mouse TLR3. I can get the cells to grow up well both with and without selection antibiotics but when I collect them by scraping and then re-plate them about 90% of the cells either die or just won't attach to the new plate. I have a similar problem with the mTLR4-293Ts, but not quite as bad. I've been growing them in DMEM with 10% FBS and, glutamine, and pen/strep as Invivogen suggests. Does anyone have experience with these cells or have any suggestions to get them to plate nicely?
I've worked with the HEK-Blue cells from InvivoGen, I believe these cells are the same except the HEK-Blue cells also express Secreted Embryonic Alkaline Phosphatases (SEAP). Personally I haven't worked with mTLR3 cells (I used hTLR4 and hNOD2) but I've never encountered dying cells to the amount you're experiencing. What may be the problem is the scraping.
What I do is first remove the spent Growth Medium, then wash once with warm PBS (37 C), remove the PBS and then add a fixed amount (say 5 mL for a T75 tissue culture flask) of warm PBS, incubate the cells in the warm PBS for a few minutes and finally detach by tapping. I found that the cells still cluster together after detaching so to remove these clusters I pipette up and down for a good while and check under the microscope if they all look detached. This seems to work for these cells.
You may also want to keep an eye on the passage number. I tend to see that at passage numbers around 15 the cells start to die and/or not adhere. Another possibility is that the cells may have lost their transfection (namely the antibiotic resistance gene) and so the selective antibiotics are starting to kill the cells.
I recommened contacting InvivoGen, they have really good customer support, and are always ready to help.
Hope this helps. And I apologize for the sloppy writing, I haven't had my coffee yet!
Cheers,
Laszlo
First make sure you are growing them on tissue culture-grade flask coated with poly lysine as it positively charge and will thus interact with negatively charge cell surface thereby ensuring adherence, alternative increase FBS to 15% and let them grow for a week without changing media, I have a similar problem with MDCK
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