1. 2 - 10 ug/mL in PBS or bicarbonate

2. Non TC polystyrene may be better than TC one. Just try.

1. Coat 5 ug/ml CD3 antibody overnight at 4 degrees or for 2 hrs in incubator (37 degrees) in PBS. For 96 well, I use 200 ul/well volume.

2. TC treated plates work better in my hands since it is polystyrene coated that helps in cells adhesion.

3. Always use flat bottom plate for coating CD3 antibody because you want CD3 antibody to be coated for a strong TCR activation.

4. CD28 should be in soluble form, since co-stimulation should not have to be stagnant (like CD3).

Hope this helps. Good luck!

I used to do 3ug/ml CD3 antibody overnight at 4 degrees or for 2~3 hrs in incubator (37degrees) in PBS. For 96 flat-bottom wells, I used 100ul/well volume.

There is currently no generally accepted best practice on this issue. This may require experimenters to explore through experiments to obtain the best method suitable for their own experiments. The following methods are for reference only. Dilute the anti-CD3 antibody with sterile PBS (at a concentration of 1 µg/mL), then coat the wells with anti-CD3 antibody. Place the culture plate in a 5% CO2 incubator and incubate at 37 °C for 2 h. Isolate PBMCs from whole blood or lymphoid tissue under sterile conditions. Resuspend cells in complete RPMI 1640 medium to a concentration of 3 x 106 cells/mL. Transfer the cell solution to a conical tube, add 5 µg/mL anti-CD28 to the conical tube, and place it in the incubator (room temperature). After 2 hours, remove the well plate from the incubator. Aspirate the anti-CD3 solution and discard. Do not wash the culture plate. Then distribute the cell solution evenly to the wells treated with anti-CD3 on the well plate.

Carlos Moreno what concentration of CD3 & CD28 that works for you?