Hi,
I am planning on stimulating T cells with plate bound anti-CD3 and anti-CD28 I know I want to use a concentration of 1ug/ml but I’m having issues finding a protocol to do this. What do I use as a buffer for coating? what do I wash with? how long should I incubate the plate for? and at what temp?
I’m sure that lack of protocols is due to its common use in labs and therefore no need to publish it but my lab has previously used the beads rather than coated plates and therefore I have no source of this protocol near by.
You’re right; For plate-bound anti-CD3 and anti-CD28 stimulation is widely used but not always explained in detailed in protocols.
Here’s a basic step-by-step guide based on standard practices:
Plate Coating Protocol for Anti-CD3 and Anti-CD28
Materials Needed: Anti-CD3 (e.g., OKT3), Anti-CD28, PBS (Phosphate-Buffered Saline) or carbonate-bicarbonate coating buffer (0.1M, pH 9.6),
Sterile tissue culture-treated 96- or 24-well plates, Sterile PBS for washing
Protocol for it:
1st step is Coating the Plate
Dilute anti-CD3 and anti-CD28 in PBS or carbonate-bicarbonate buffer to a final concentration of 1 µg/mL each.
Add 50-100 µL per well (96-well plate) or ~500 µL per well (24-well plate).
Incubate the plate overnight at 4°C or 2 hours at 37°C (if short on time).
Washing
2nd is to Remove the antibody solution.
Wash 2-3 times with sterile PBS to remove unbound antibodies.
If desired, block with 1% BSA in PBS for 30 minutes at 37°C to reduce non-specific binding (optional but recommended).
3rd step is - Cell Seeding & Stimulation
Wash again with PBS before adding cells.
Seed T cells in appropriate culture medium (e.g., RPMI 1640 + 10% FBS) at desired density (typically 0.5-1 × 10⁶ cells/mL).
Incubate at 37°C, 5% CO₂ for 24-72 hours, depending on your experiment.
This should get your T cell activation working smoothly!