Transfecting 293 in serum free medium - problems with growth (Dec/13/2007 )
Hello,
We currently use 293 HEK tet-on cells from Cambrex to test constructs in. We have a angiopoietin construct that we want to test with this system. Since Ang is secreted, we need to collect the medium, IP, and then do a western. So, we are trying to put our 293's into serum free medium to grow since the serum effects our Western. However, when we do this, our cells won't attach...even to collagen plates.
Is there something else I'm missing here or is serum in the medium that crucial to help the cells attach?
Many thanks in advance!
What type of medium did you use? Have you tried opti-MEM?
Right now we are using the Pro293-a from Lonza...supposed to be for attached cells, but no luck getting them to sit down on the plate again...
Right now we are using the Pro293-a from Lonza...supposed to be for attached cells, but no luck getting them to sit down on the plate again...
I have not used that one. Try it and I hope your problem will be gone.
Could you plate the cells in serum containing media and in 2-3 hrs when the cells are attached to the plate, change the media to the one for your experiments.
Also you could try using B27 supplements instead of serum. We use it for neurons to avoid using serum.
Yep.. you must have serum in order for cells to attach. I'm not sure exactly what the serum supplies but I tried to put my cells under serum starvation during splitting and they never attached (CHO cells). You can split, allow them to attach and then change the media. I would recommend you keep at least 0.1% serum though, to avoid apoptosis. Hopefully this is a small enough concentration that it won't interfere with the western.
bump
Some protocols suggest a gradual adoptation process moving cells from serum-containing to serum free medium. I wonder if you have done the same. It could take a while, weeks, I believe, to do the step down process.
Hey,
the thing is quite easy to handle. Split cells as usual with serum and grow them to 95 % confluency, e.g. in DMEM (Low Glucose; 8 % FCS; 2 mM Gln; 1 % PS. From then on you can set them serum free (e.g. DMEM) by carefully washing the cells once with prewarmed PBS and the cultureing for 12 h or 24 hours in serum free medium. The cells cn deal very well with serum free conditions once they have grown in serum!!!! you can make also a time course, like collecting the supernatant after 6 h, 12 or 24 hours. This will defintely work!!!!! Hope I was ble to help you!!!! We have a great expertise in 293 cell... Feel free to contact me for further questions! 
Greetz
Kai
if you remove the serum, you starve the cells.
what i do is, plate them for the exeriment in 10%FBS, and the day before replace the media with serum free.
cells need to eat before they attach.
V