Resuspend cells at 106-107 cells/ml with cold serum-free media.
Add CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) #92511 to cells at 1:200 to reach a final concentration of 10 µg/ml.
Optional: Add Human CD28 Activating (CD28.2) Mouse mAb (Low Endotoxin, Azide-free) #91920 to cells at 1:200 to reach a final concentration of 10 µg/ml.
Keep cells with agonist antibody on ice for 30 minutes.
Collect cells by centrifugation.
Aspirate the supernatant.
Resuspend cells at 106-107 cells/ml with cold serum-free media and keep on ice.
Add Goat Anti-Mouse Kappa Light Chain, F(ab’)2 Antibody (Low Endotoxin, Azide-free) #75861 secondary antibody to cells at 1:50 to reach a final concentration of 10 µg/ml if only CD3ε (OKT3) was used; add at 1:25 to reach a final concentration of 20 µg/ml if both CD3ε (OKT3) and CD28 (CD28.2) were used.
Keep cells with secondary antibody on ice for 15 minutes.
To initiate stimulation, transfer cells to 37 °C water bath for desired stimulation time.
To terminate stimulation, follow the cell preparation protocol recommended for the desired analysis method.
C. Stimulation Protocol: Plate-based
Prepare a 10 µg/ml solution of CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) #92511 in sterile PBS. Prepare a sufficient volume to cover the bottom of all the wells to be used for the stimulated T cell condition (approximately 50 µl for 96-well plates, 1 ml for 6-well plates).
Add the diluted CD3ε (OKT3) antibody solution to the wells chosen for treatment. Add equivalent volume of sterile PBS to the wells chosen as controls for treatment.
Cover the plate and incubate at 37 °C for a minimum of 2 hrs. The plate may be allowed to incubate overnight if desired.
Prepare cells as needed to generate a cell suspension of roughly 1 x 106 cells/ml in a sufficient volume of complete media (100 µl per well for 96-well plates, 2 ml for 6-well plates). Optimal cell density may be determined through experimentation with cells of interest. For T cell expansion, add Human Interleukin-2 (hIL-2) #8907 to the cell suspension to yield a final concentration of 20 ng/ml.
Aspirate the CD3ε (OKT3) antibody solution from the wells, and rinse wells 2x with excess sterile PBS to remove unbound antibody.
Add cell suspension to all wells, at volumes specified in step 4 or as determined by experimentation.
Add Human CD28 Activating (CD28.2) Mouse mAb (Low Endotoxin, Azide-free) #91920 to yield a concentration of 2 µg/ml. Pre-dilute CD28 (CD28.2) antibody in complete media if needed. CD28 antibody may also be added to a portion of control wells if desired.
Incubate cells for desired time in a humidified incubator (37 °C, 5% CO2). Add fresh media with IL-2 as needed for T cell expansion.
Harvest cells and proceed with immunostaining or analysis as desired.
Protocol Id: 2450
The Human Anti-CD3/CD28 T Cell Activation Kit provides the necessary antibodies to induce human T cell activation and proliferation using the soluble stimulation protocol.
Specificity / Sensitivity
The Human Anti-CD3/CD28 T Cell Activation Kit is used for the stimulation of human T cells following the soluble stimulation protocol.
T cells are activated by signaling through ITAM (immunoreceptor tyrosine-based activation motif)-containing CD3 signaling chains that associate with the T cell receptor (1). Co-stimulation through CD28 on T cells provides an additional signal required for effective T cell activation (2). Signaling through both CD3 and CD28 can be initiated using the agonist antibodies Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) and Human CD28 Activating (CD28.2) Mouse mAb (Low Endotoxin, Azide-free), respectively. If the agonist antibodies are used in the soluble stimulation protocol, cross-linking of their Fc domains using Goat Anti-Mouse Kappa Light Chain, F(ab')2 Antibody (Low Endotoxin, Azide-free) is required for optimal stimulation.