As we failed to see the boosted level of certain cytokines in activated mouse T cell, and also see bad cell status such as shrinked cell membrane and no cell aggregation upon antibody stimulation, is it possibly caused by the complement in the serum as I forget to inactivat it? Thanks.
Hi Sophie Gong
What you have said about serum inactivation is, highly, probable the cause of your results!
Regards!
I am not sure. We never heat inactivate our serum when doing mouse T cell activation (in our case using irradiated splenocytes + peptide), and the cells become activated very strongly. But maybe this depends on the serum provider/batch?
Well, have got the poor result that EL4 cells were still not boosted with cytokine secretion in all groups with 0, 1, 5, 10ug/ml anti-CD3 coating and anti-CD28/TGFb1/IL-6 in suspension, even after serum is inactivated. So if we temporally believe it is better to have it inactivated, what's the possible reason for the failure of T cell activation with significant cytokine secretion? I presume 2 other reasons: 1) poor coating of CD3 antibody to plate surface as I used tissue cultured treated ones (more hydrophilic), will it be better if use untreated one as it will increase passive binding of antibodies to the surface? 2) can I directly change to DynaBeads of anti-CD3/anti-CD28 and directly throw it into the medium for higher chance of success? But why still many people are using traditional antibody coating protocols? Will there be some pro and con between two methods?
Thank you, if any one can make some comment...
Hi Sophie, using non-treated tissue culture plates for antibody coating is crucial! I have found this using EL4 cells and anti-CD3 antibody coating, so the setup is very comparable. In my hands, coating works best when done O/N at 4 degrees in 0.1 M sodium bicarbonate buffer, pH 9.2. Worth a try.
Hi Sophie,
I've had great success with dynabeads in a 1:1 or 2:1 ratio of beads to primary mouse CD4 T cells. I just spin them down, resuspend in medium and directly add to my cells. I've used tissue culture treated plates for this. Hope it helps.
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