Introduction

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Mature T cells recognize and respond to the antigen/MHC complex through their antigen-specific receptors (TCR). The most immediate consequence of TCR activation is the initiation of signaling pathways including induction of specific protein tyrosine kinases (PTKs), breakdown of phosphatidylinositol 4,5-biphosphate (PIP2), activation of protein kinase C (PKC) and elevation of intracellular calcium ion concentration. These early events are transmitted to the nucleus and result in

• 1. clonal expansion of T cells
  2. upregulation of activation markers on the cell surface
  3. differentiation into effector cells
  4. induction of cytotoxicity or cytokine secretion
  5. induction of apoptosis

One of the most common ways to assess T cell activation is to measure T cell proliferation upon in vitro stimulation of T cells via antigen or agonistic antibodies to TCR.

This protocol is written as a starting point for examining in vitro proliferation of mouse splenic T-cells and human peripheral T cells stimulated via CD3. Critical parameters include cell density, antibody titer and activation kinetics.

Materials

• 1X sterile PBS
• Anti-mouse CD3e, Clone 145-2C11 (Functional Grade, Cat. No. 16-0031 , or Purified, Cat. No. 14-0031 )
• Complete RPMI-1640
• Sterile single-cell suspension of mouse spleen or lymph nodes
• 96-well flat-bottom microtiter plates with lids (Costar Cat. No. 3596)
• MTT Buffer
• MTT Lysing Solution
• 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

• 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

Method

Antibody Coating of the Assay Plate Microwells:

1. Prepare a 5-10µg/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 0.5-0.1µg/ml 145-2C11 antibody solution can be used.
2. Dispense 50µl of the antibody solution to each well of the 96-well assay plate. For the control unstimulated wells, add 50µl of sterile PBS.
3. Tightly cover the plate with Parafilm^TM 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.
4. Just before adding cells, remove the 50µl antibody solution with a multichannel pipettor.
5. Rinse each well with 200µl 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 conditions. Follow the red cell lysis protocol to remove red cells.
2. 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.
3. After washing the wells with PBS (step 6 above), add 200µl 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 1-4µg/ml of culture medium.
4. 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.
5. Add 20µl of the MTT buffer to each well and put back in the incubator for 4 hours.
6. Add 50µl of the MTT Lysis Solution to each well, vortex gently and incubate overnight.
7. Read the plate at 570nm the next day.
8. Calculate the mean and standard errors and graph the data.

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Human Protocol: Stimulation of human peripheral blood mononuclear cells with anti-human CD3 monoclonal antibody; MTT assay for detection of cellular proliferation.

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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.

Materials

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

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 1-2x10⁶ /ml of complete RPMI. Note: This density of PBMC gives a good response. If experimental conditions require, a titration of cell densities (2-3x10⁶ /ml to 10⁵ /ml) should be performed for optimization.
2. Add 100µl of the cell suspension to each well. For each condition, use triplicate wells.
3. Add soluble antibody in 100µl 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 10µg/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 response; however, this incubation time can also be optimized for specific experimental conditions.
6. Add 20µl of the MTT buffer to each well and put back in the incubator for 4 hours.
7. Add 50µl of the MTT Lysis Solution to each well, vortex gently and incubate overnight.
8. Read the plate at 570nm the next day.
9. Calculate the mean and standard errors and graph the data.

Buffers & Media

• Complete RPMI-1640:
  • 900ml RPMI-1640 (Hyclone Cat. No. SH30027.02)
  • 100ml FBS (Hyclone Cat. No. SH30151.03) Heat inactivated (10% final)
  • 1ml 2-mercaptoethanol (Gibco BRL Cat. No. 21985-023)
  • 10ml L-Glutamine (Hyclone Cat. No. SH30034.01)
  • Antibiotic cocktail (optional)
• MTT Buffer:
  • MTT (Sigma Cat. No. M5655) 5mg/ml stock solution in 1X PBS - Keep protected from light.
• MTT Lysing Solution:
  • 20% SDS 50% DMF