Protocol of Cell Activation for Flow Cytometry

General activation protocols often use pharmacological agents and antibodies, making ideal to determine immune competence, marker upregulation, cytokine release and proliferation by flow cytometry. Protocols provided here include the following parts. Refer to the corresponding sections according to your experimental requirements.

Contents:

• Protocol A: Unprimed T Cell Activation - Pharmacologic Method
• Protocol B: Unprimed T Cell Activation - Antibody Stimulation Method
• Protocol C: Primed T Cell Activation - Antigen Presenting Cell Co-culture

Protocol A: Unprimed T Cell Activation -Pharmacologic Method

Protocol A provides a common procedure for activating T cells prior to staining for intracellular cytokines, activation markers or cell proliferation using pharmacologic means. These include agents such as phorbol 12-myristate13-acetate (PMA) and ionomycin, phytohaemagglutinin (PHA) and concanavalin A (ConA). All blood samples must be collected into heparin anticoagulant.

Reagents

• PBS
• Cell media
• Stimulation reagents

Methods

1. Isolate PBMC using the appropriate methods. Refer to Protocol of Cell Preparation for Flow Cytometry.
2. Resuspend in the appropriate media and adjust to 1 x 10⁶ cells/mL.
3. Add the pharmacological agent to each well. Refer to the above table for reagents and corresponding final concentrations. This will differ according to the species and origins of your cells and the experimental requirements. Add an equivalent amount of PBS or media to a tube to act as an unstimulated control.
4. Add 1x 10⁵ cells and incubate cells in a humidified 37°C, 5% CO₂ incubator for 6-72 h depending on your experimental needs.
5. Harvest cells and perform surface or intracellular staining as required.
(Refer to Protocol of Intracellular Staining Flow Cytometry for more details.)
6. Acquire by flow cytometry.

Protocol B: Unprimed T Cell Activation - Antibody Stimulation Method

Protocol B provides a common procedure for activating T cells prior to staining for intracellular cytokines, activation markers or cell proliferation using anti-CD3 and anti-CD28 antibodies. Blood samples must be collected in heparin anticoagulant.

Reagents

• PBS
• Cell media
• Anti-CD3 and CD28 antibodies

Methods

1. Prepare a 5-10 µg/mL solution of anti-CD3 in sterile PBS.
2. Add 50-100 µL to each well of a 96 well plate, seal and incubate overnight at 4°C or 2 h at 37°C. For the unstimulated control, add 50-100 µL of sterile PBS.
3. Remove supernatant and wash the plate with PBS to remove unbound antibody.
4. Isolate PBMC using the appropriate methods. Refer to Protocol of Cell Preparation for Flow Cytometry.
5. Resuspend in the appropriate media and adjust to 1 x 10⁶ cells/mL.
6. Add 1x 10⁵cells to each well.
7. Add CD28 antibody at 1-5 µg/mL and incubate cells in a humidified 37°C, 5% CO₂ incubator for 6-96 h as required.
8. Harvest cells and perform surface or intracellular staining as required.
(Refer to Protocol of Intracellular Staining Flow Cytometry for more details.)
9. Acquire by flow cytometry.

Protocol C: Primed T Cell Activation - Antigen Presenting Cell Co-culture

Protocol C provides a common procedure for activating T cells prior to staining for intracellular cytokines, activation markers or cell proliferation using dendritic cells (DCs) that have been pulsed with antigen. Other types of antigen presenting cells may require different amounts of antigen and incubation times.

Reagents

• PBS
• Cell media
• Lipopolysaccharide (LPS)

Methods

1. Prepare DCs from an appropriate source.
2. Incubate DCs with antigen (50-200 ug/mL) overnight in the presence of LPS (100 ng/mL).
(Antigen should be titrated to obtain reproducible results. Non-pulsed DCs should be included as a negative control.)
3. Wash the DCs twice in cell culture media. Count and resuspend at 1 x 10⁶ /mL.
4. Isolate T cells and resuspend in media at 1 x 10⁶ cells/mL.
5. Co-culture the DCs and T cells at increasing ratios. For instance, DC: T cell = 1:1, 1:5 and 1:10 to obtain a range of stimulation.
6. Incubate cells in a humidified 37°C, 5% CO₂ incubator for 6-96 h as required.
7. Harvest cells and perform surface or intracellular staining as required.
(See Protocol of Intracellular Staining Flow Cytometry for more details.)
8. Acquire by flow cytometry.