What You Need

Materials

• 1X sterile PBS
• Anti-mouse CD3e, 145-2C11:
  • Functional Grade, Cat. No. 16-0031 , or
  • Purified, Cat. No. 14-0031
• Supplemented RPMI-1640 < click here > for recipe
• Sterile single-cell suspension of mouse spleen or lymph nodes
• 96-well flat-bottom microtiter plates with lids (Costar Cat. No. 3596)
• MTT Buffer < click here > for recipe
• MTT Lysing Solution < click here > for recipe
• Concanavalin A, optional (ConA, Sigma Cat. No. C5275)

Instruments

• Pipettes and pipettors, Multichannel pipettor
• Centrifuge
• 37°C, CO₂ incubator
• 96-well micro test spectrophotometer

Experiment Duration

• 20 minutes antibody preparation for coating wells
• 20 minutes preparation of spleen single cell suspension
• 20 minutes to set up the assay
• 2-4 days incubation

Method

Antibody Coating of the Assay Plate

1. Prepare a 5-10 µg/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) 2.6 ml 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 co-stimulation studies using antibodies to other antigens, a suboptimal activation with anti-CD3 may be required. To achieve suboptimal activation via anti-CD3, a 0.5-0.1 µg/ml 145-2C11 antibody solution can be used.
2. Dispense 50 µl of the antibody solution in each well of the 96-well assay plate. For the control unstimulated wells, add 50 µl of sterile PBS.
3. Tightly cover the plate with Parafilm to avoid sample evaporation and incubate at 37°C for 2 hours or alternatively prepare the plate one day in advance and keep at 4°C overnight.
4. Just before adding cells, remove the 50 µl antibody solution with a multichannel pipettor.
5. Rinse each well with 200 µl of sterile PBS and discard PBS.
6. Repeat step 5 to remove all unbound antibody from each well.

Addition of Cells

1. Harvest spleen and prepare a single cell suspension under sterile condition. Follow the red cell lysis protocol to remove red cells. < click here > for RBC lysis protocol.
2. Count cells and resuspend in complete RPMI-1640 at 10⁶ /ml.
   Note: This concentration of spleen cells gives a good response. If experimental conditions require, a titration of cell concentrations (2-3 x 10⁶ /ml to 10⁵ /ml) should be performed for optimization.
3. After washing the wells with PBS (step 6 above), add 200 µl 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 1-4 µg ConA per ml of culture medium.
4. Incubate for 2-4 days.
   Note: Proliferation of cells between days 2 and 4 gives a good signal; however, this incubation time can also be optimized for specific experimental conditions.
5. Add 20 µl of the MTT buffer to each well and put back in the incubator for 4 hours.
6. Add 50 µl of the MTT Lysis Solution to each well, vortex gently and incubate overnight
7. Read the plate at 570 nm the next day.
8. Calculate the mean and standard errors and graph the data.

Human PBMCs can be activated in vitro by soluble anti-human CD3 antibodies. We have performed titration studies with these antibodies and established the following protocol for stimulation of PBMC.

What You Need

Materials

• 1X sterile PBS
• Anti-human CD3:
  • OKT3, Functional Grade Cat. No. 16-0037 , or
  • HIT3a, Functional Grade Cat. No. 16-0039
• Supplemented RPMI-1640 < click here > for recipe
• Sterile PBMC
• 96-well flat-bottom microtiter plates with lids (Costar Cat. No. 3596)
• MTT Buffer < click here > for recipe
• MTT Lysing Solution < click here > for recipe

Instruments

• Pipettes and pipettors, Multichannel pipettor
• Centrifuge
• 37°C, CO₂ incubator
• 96-well micro test spectrophotometer

Experiment Duration

• 30 minutes preparation of PBMC
• 20 minutes to set up the assay
• 2-4 days incubation

Method

1. Prepare PBMC and resuspend the cells at 1-2 x 10⁶ /ml of supplemented RPMI.
   Note: This concentration of PBMC gives a good response. If experimental conditions require, a titration of cell concentrations (2-3 x 10⁶ /ml to 10⁵ /ml) should be performed for optimization.
2. Add 100 µl of the cell suspension to each well. For each condition, use triplicate wells.
3. Add soluble antibody in 100 µl to each well. Titrate antibodies for optimal performance in the assay conditions used. If isolated T cells are to be used in proliferation assays, we recommend using these antibodies at 10 µg/ml immobilized on plastic (i.e. bound to wells of 96-well assay plates).
4. Place in a humidified 37°C, 5% CO₂ incubator.
5. Incubate for 2-4 days.
   Note: Proliferation of cells between day 2 and 4 gives a good signal; however, this incubation time can also be optimized for specific experimental conditions.
6. Add 20 µl of the MTT buffer to each well and put back in the incubator for 4 hours.
7. Add 50 µl of the MTT Lysis Solution to each well, vortex gently and incubate overnight.
8. Read the plate at 570 nm the next day.
9. Calculate the mean and standard errors and graph the data.

Supplemented RPMI-1640

• 900 ml RPMI-1640 (Hyclone Cat. No. SH30027.02)
• 100 ml FBS (Hyclone Cat. No. SH30151.03) Heat inactivated (10% final)
• 1 ml 2-mercaptoethanol (GibcoBRL Cat. No. 21985-023)
• 10 ml L-Glutamine (Hyclone Cat. No. SH30034.01)
• Antibiotic cocktail (optional)

MTT Buffer

• MTT (Sigma Cat. No. M5655) make a 5 mg/ml stock solution in 1X PBS - Keep protected from light.

MTT Lysing Solution

• 20% SDS 50% DMF