I have been trying a protocol for T cell activation and differentiation using murine splenocytes in vitro, but I have been getting a lot of dead cells as indicated by flow cytometry. I am coating a 96-well round bottom cell culture plate with 5ug/mL CD3 plus 2ug/mL CD28 antibodies in PBS(--), at 100uL per well. The plate is then stored overnight at 4C. I had been making my CD3/CD28 solution as 5ug/mL CD3 plus 2ug/mL CD28 in 1mL PBS and transferring 100uL of this into each well, which would alter the final concentration that is actually coated onto the plate.
1) Is there an optimal range for each plate-bound CD3 and CD28 antibodies that should be added to the wells? I have not found consistent information for this in online protocols.
2) I have been using PBS without Mg/Ca, but one protocol uses PBS with Mg/Ca. Would this difference in PBS affect antibody binding to the plate?
I am wondering if the final concentrations of CD3/CD28 I have used are not enough for T cell stimulation and also wondering if these antibodies are actually plate-bound. Any help is much appreciated!
Hello Cristina Arenaz
Splenic T cells are stimulated with immobilized anti-CD3 and solubilized anti-CD28 antibodies in complete RPMI 1640 medium supplemented with 10%FBS, 50uM beta-mercaptoethanol, 1mM sodium pyruvate and 2mM L-glutamine in 96- well plate for 40-72 hours.
Most T cells will require 3 days to divide. These cells will clump but will not be attached to the plate. Harvest the cells using the cell scrapper.
The optimum concentration of anti-CD3 ranges from 1-3ug/ml and anti-CD28 ranges from 3-5ug/ml. You may add recombinant IL-2 (100ng) as it will stimulate the growth and differentiation of cytotoxic T cells.
Answer 1.
You should use anti-CD3 in the range of 1-3ug/ml. (1ug/ml anti-CD3 is preferable). Use solubilized anti-CD28 in the range of 3-5ug/ml, (5ug/ml anti-CD28 is preferable). You may coat the wells of 96-well plate only with anti-CD3 at 1ug/ml in sterile PBS. You could use anti-CD28 at 5ug/ml in the medium containing the cells.
After incubating the plate with anti-CD3 for 2 hours at 37 degree C or overnight at 4 degree C, you may aspirate the anti-CD3 solution. Do not wash the plate. Add the activated cell suspension in the anti-CD3 treated wells. Incubate the plate for 72 hours.
Answer 2.
Use PBS without Ca2+/Mg2+.
Hope this protocol helps!
Good Luck!
Hi,
In addition to the answers and explanations above, I would recommend two new things that worked in my hands:
1. Use flat bottom for coating instead of round bottom plate.
2. I do 3 hours incubation of the plate in incubator (37 degrees) after coating with anti-CD3 instead of overnight incubation.
Hope this helps.
Good luck!