| Please <click here> for Mouse T-cell Activation Protocol (Protocol A)
Please <click here> for Human T-cell Activation Protocol (Protocol B) Protocol A: Stimulation of mouse peripheral T cells with plate-bound 145-2C11 monoclonal antibody; MTT assay for detection of cellular proliferation. This protocol is written as a starting point for examining in vitro proliferation of splenic T-cells stimulated via CD3. All the steps where optimization by the user can be achieved for meaningful results have been highlighted. Briefly, these include titration of responding cells, titration of the activating reagents and the optimal culture period for the best activation response. 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 degrees 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 Degree of difficulty: Easy Method: ANTIBODY COATING OF THE ASSAY PLATE: 1) Prepare a 5-10 ug/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, if desired, a pilot experiment to determine efficacy of other concentrations of this antibody to induce cellular activation can be performed. In order to achieve suboptimal concentration of anti-CD3 in co-stimulation assays using antibodies to other antigens, a 0.5-0.1 ug/ml 145-2C11 antibody solution can be used. 2) Dispense 50 ul of the antibody solution in each well of the 96-well assay plate. For the control unstimulated wells, add 50 ul of sterile PBS. 3) Tightly cover the plate with Parafilm to avoid sample evaporation and incubate at 37 degrees C for 2 hours or alternatively prepare the plate one day in advance and keep at 4 degrees C overnight. 4) Just before adding cells, remove the 50 ul antibody solution with a multichannel pipettor. 5) Rinse each well with 200 ul 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. for protocol 2) Count cells and resuspend in complete RPMI-1640 at 10(6)/ml. Note: This concentration of spleen cells gives a good response. If experimental conditions require, a titration of cell concentrations (2-3 x 10(6)/ml to 10(5)/ml) should be performed for optimization. 3) After washing the wells with PBS (step 6 above), add 200 ul of the cell suspension to each well and place in a humidified 37 degrees C, 5% CO2 incubator. Note: For an additional stimulation control, incubate cells in 3 wells with Concanavalin A (ConA, Sigma cat.no. C5275) at 1-4 ug 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 ul of the MTT buffer to each well and put back in the incubator for 4 hours. 6) Add 50 ul of the MTT Lysis Solution to each well, vortex gently. 7) Read the plate at 570 nm the next day. 8) Calculate means and standard errors and graph the data.
Protocol B: Stimulation of human peripheral blood mononuclear cells with anti-human CD3 monoclonal antibody; MTT assay for detection of cellular proliferation. Human PBMCs can be activated in vitro by soluble anti-human CD3 antibodies such as UCHT1 and HIT3a. We have performed titration studies on both of these antibodies and established the following protocol for stimulation of PBMC. If isolated T cells are to be used in proliferation assays, we recommend using these antibodies at 10 ug/ml immobilized on plastic (i.e. bound to wells of 96-well assay plates). What you need: *Materials: -1X sterile PBS -Anti-human CD3: UCHT1, Functional Grade cat.no. 16-0038 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 degrees 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 Degree of difficulty: Easy Method: 1) Prepare PBMC and resuspend the cells at 1-2 x 10(6)/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 10(6)/ml to 10(5)/ml) should be performed for optimization. 2) Add 100 ul of the cell suspension to each well. For each condition, use triplicate wells. 3) If using UCHT1, add soluble antibody in 100 ul to each well at a final concentration of 1-10 ug/ml. If using HIT3a add soluble antibody in 100 ul to each well at a final concentration of 0.01-0.1 ug/ml. 4) Place in a humidified 37 degrees 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 ul of the MTT buffer to each well and put back in the incubator for 4 hours. 7) Add 50 ul of the MTT Lysis Solution to each well, incubate overnight. 8) Read the plate at 570 nm. 9) Calculate means and standard errors and graph the data. Buffers & Media: *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 | 