Mouse Protocol: Stimulation of mouse peripheral T cells with plate-bound 145-2C11 monoclonal antibody;
MTT assay for detection of cellular proliferation.
1X sterile PBS
Anti-mouse CD3e, Clone 145-2C11 (Functional Grade, Cat. No. 16-0031, or Purified, Cat. No. 14-0031)
Sterile single-cell suspension of mouse spleen or lymph nodes
96-well flat-bottom microtiter plates with lids (Costar Cat. No. 3596)
MTT Lysing Solution
Concanavalin A, optional (ConA, Sigma Cat. No. C5275)
Pipettes and pipettors, Multichannel pipettor
37°C, CO2 incubator
96-well micro test spectrophotometer
2 hours to coat antibody to flask or plate
20 minutes preparation of spleen single cell suspension
20 minutes to set up the assay
2-4 days incubation
Antibody Coating of the Assay Plate Microwells:
Prepare a 5-10g/ml solution of anti-CD3e (145-2C11) in sterile PBS. Calculate the number of wells required for each experimental condition and consider triplicate samples for each condition. For example, to coat one-half plate (48 wells) 2.6ml of antibody solution is required. Note: We have performed titration studies and found these concentrations of 145-2C11 to induce a maximal response. However, a pilot experiment to determine efficacy of other concentrations of this antibody to induce cellular activation can be performed. For costimulation studies using antibodies to other antigens, a suboptimal activation with anti-CD3 may be required. To achieve suboptimal activation via anti-CD3, a 0.5-0.1g/ml 145-2C11 antibody solution can be used.
Dispense 50l of the antibody solution to each well of the 96-well assay plate. For the control unstimulated wells, add 50l of sterile PBS.
Tightly cover the plate with ParafilmTM to avoid sample evaporation and incubate at 37°C for 2 hours or prepare the plate one day in advance and keep at 4°C overnight.
Just before adding cells, remove the 50l antibody solution with a multichannel pipettor.
Rinse each well with 200l of sterile PBS and discard PBS.
Repeat step 5 to remove all unbound antibody from each well.
Addition of Cells:
Harvest spleen and prepare a single cell suspension under sterile conditions. Follow the red cell lysis protocol to remove red cells.
Count cells and resuspend in complete RPMI-1640 at 106/ml. Note: This density of spleen cells gives a good response. If experimental conditions require, a titration of cell densities (2-3x106/ml to 105/ml) should be performed for optimization.
After washing the wells with PBS (step 6 above), add 200l of the cell suspension to each well and place in a humidified 37°C, 5% CO2 incubator. Note: For an additional stimulation control, incubate cells in 3 wells with Concanavalin A at 1-4g/ml of culture medium.
Incubate for 2-4 days. Note: Proliferation of cells between days 2 and 4 gives a good response; however, this incubation time can also be optimized for specific experimental conditions.
Add 20l of the MTT buffer to each well and put back in the incubator for 4 hours.
Add 50l of the MTT Lysis Solution to each well, vortex gently and incubate overnight.
Read the plate at 570nm the next day.
Calculate the mean and standard errors and graph the data.