In vitro T cell activation assay of mouse spleen T cells and human PBMCs

preface

Mature T cells recognize and react with the antigen-MHC complex by TCR. The most immediate consequence of TCR activation is the initiation of signaling pathways including induction of specific protein tyrosine kinases (PTKs), PIP2 breakdown, PKC activation, and elevation of intracellular calcium ion concentrations. These early events are passed on to the nucleus and have many effects:

1. Amplification of T cell clones

2. Upregulation of cell surface activation markers

3, differentiation into effector cells

4. Inducing cytotoxic or cytokine secretion

5. Inducing apoptosis

The most common method of in vitro stimulation to detect T cell proliferation is to activate T cells with TCR antigens or competitive antibodies.

This protocol is a basic method for in vitro stimulation of mouse spleen T cells and human peripheral blood T cells by CD3. Key parameters include cell density, antibody titer and activation kinetics.

First, the mouse:

Stimulation of mouse T cells with coated 145-2C11 mAb; cell proliferation by MTT assay

material

1X sterile PBS

Anti-mouse CD3e, Clone 145-2C11 (Functional Grade, eBioscience Cat. No. 16-0031, or Purified, eBioscience Cat. No. 14-0031)

RPMI-1640 complete medium

Sterile mouse spleen or lymph node single cell suspension

96 hole flat bottom with microplate

MTT Buffer

MTT lysate

Concanavalin A, optional (ConA, Sigma Cat. No. C5275)

Instrument

Micropipette or gun

Centrifuge

37 ° C, CO2 incubator

96-well plate reader

Experimental time

2 hours coated antibody

Preparation of spleen single cell suspension in 20 minutes

20 minute analysis setup

2-4 days incubation

Experimental procedure

Antibody coated to microplate:

1. Prepare 5-10 μg/ml anti-CD3e (145-2C11) antibody solution in sterile PBS, calculate the amount of antibody required, and perform duplicate wells for each condition or sample. For example, a half board is required to require 2.6 ml of antibody solution. The concentration of CD3 antibodies can be reduced if co-stimulated with other antibodies.

2. Add 50 μl of antibody to each well and add 50 μl of sterile PBS to the blank control wells.

3. Seal the plate with ParafilmTM membrane and incubate at 37 °C for 2 hours or 4 °C overnight.

4. Absorb the antibody solution in the well before adding the cells.

5. After washing each well with 200 μl of sterile PBS, discard the PBS.

6. Repeat the wash once to remove all unbound antibodies.

Add cells:

1. Take the spleen and prepare a single cell suspension aseptically. Hemolysis removes red blood cells.

2. Count the cells and resuspend in RPMI-1640 complete medium at a concentration of 106/ml. A concentration of 2-3 x 106 / ml ~ 105 / ml can be selected according to the experimental needs.

3. After washing the plate, add 200 μl of cell suspension to each well and place in a humidified 37 ° C, 5% CO2 incubator. Optional plus stimulation control: ConA 1-4μg/ml incubation after stimulation.

4. Incubate for 2-4 days. Can be optimized according to specific experiments.

5. Add 20 μl of MTT buffer to each well and place it back in the incubator for 4 hours.

6. Add 50 μl of MTT Lysate to each well, shake gently and incubate overnight.

7. Read the board at 570nm the next day.

8. Calculate the mean and standard deviation and plot.

Second, people:

Human peripheral blood mononuclear cells (PBMCs) were stimulated with CD3 monoclonal antibody; cell proliferation was measured by MTT assay.

material

1X sterile PBS

Anti-human CD3:

Clone OKT3 (Functional Grade, eBioscience Cat. No. 16-0037) or Clone HIT3a (Functional Grade, eBioscience Cat. No. 16-0039)

RPMI-1640 complete medium

Sterile PBMC

96-well flat bottom with microplate

MTT Buffer

MTT lysate

Instrument

Micropipette or gun

Centrifuge

37 ° C, CO2 incubator

96-well plate reader

Experimental time

30 minutes to prepare PBMCs

20 minute setup analysis

2-4 days incubation

Experimental procedure

1. Prepare PBMCs and resuspend in RPMI complete medium at a concentration of 1-2 x 106/ml. The cell concentration can be adjusted to 2-3 x 106 / ml ~ 105 / ml according to the experiment.

2. Add 100 μl of cell suspension to each well and make duplicate wells for each sample.

3. Add 100 μl of soluble antibody to each well to optimize antibody concentration. If isolated T cells are used for proliferation analysis, it is recommended to use 10 μg/ml antibody coating for stimulation on 96-well plates.

4. Place in a humidified 37 ° C, 5% CO2 incubator.

5. Incubate for 2-4 days. Can be optimized according to specific experiments.

6. Add 20 μl of MTT buffer to each well and place it back in the incubator for 4 hours.

7. Add 50 μl of MTT Lysate to each well, shake gently and incubate overnight.

8. Read the board at 570nm the next day.

9. Calculate the mean and standard deviation and plot.

Buffer and medium

RPMI-1640 Complete Medium:

900ml RPMI-1640

Heat inactivated 100ml FBS (10% final concentration)

1ml 2-mercaptoethanol

10ml L-glutamate

Antibiotic mixture (optional)

MTT Buffer:

MTT stock solution: dissolved in 1X PBS, concentration 5mg/ml, protected from light

MTT Lysate:

20% SDS 50% DMF

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