Protocol of Cell Activation for Flow Cytometry

General activation protocols often use pharmacologicalreagents and antibodies, making ideal to determine immune competence, markerupregulation, cytokine release and proliferation by flow cytometry. Protocolsprovided here include the following parts. Refer to the corresponding sectionsaccording to your experimental requirements.

Contents:

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

Protocol A: Unprimed T Cell Activation -Pharmacologic Method

ProtocolA provides a common procedure for activating T cells prior to staining forintracellular cytokines, activation markers or cell proliferation usingpharmacologic 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. IsolatePBMC using the appropriate methods. Refer to Protocol of Cell Preparation for Flow Cytometry.
2. Resuspendin the appropriate media and adjust to 1 x 10⁶ cells/mL.
3. Addthe pharmacological agent to each well. Refer to the above table for reagentsand corresponding final concentrations. This will differ according to thespecies and origins of your cells and the experimental requirements. Add an equivalentamount of PBS or media to a tube to act as an unstimulated control.
4. Add 1x 10⁵cells and incubate cells in a humidified 37°C, 5% CO₂ incubator for6-72 h depending on your experimental needs.
5. Harvestcells and perform surface or intracellular staining as required.
(Refer to Protocol of Intracellular Staining Flow Cytometry for more details.)
6. Acquireby flow cytometry.

Protocol B: UnprimedT Cell Activation - Antibody Stimulation Method

Protocol B provides a commonprocedure for activating T cells prior to staining for intracellular cytokines,activation markers or cell proliferation using anti-CD3 and anti-CD28antibodies. Blood samples must be collected in heparin anticoagulant.

Reagents

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

Methods

1. Preparea 5-10 µg/mL solution of anti-CD3 in sterile PBS.
2. Add50-100 µL to each well of a 96 well plate, seal and incubate overnight at 4°Cor 2 h at 37°C. For the unstimulated control, add 50-100 µL of sterile PBS.
3. Removesupernatant and wash the plate with PBS to remove unbound antibody.
4. IsolatePBMC using the appropriate methods. Refer to Protocol of Cell Preparation for Flow Cytometry.
5. Resuspendin the appropriate media and adjust to 1 x 10⁶ cells/mL.
6. Add 1x 10⁵cells to each well.
7. AddCD28 antibody at 1-5 µg/mL and incubate cells in a humidified 37°C, 5% CO₂ incubator for 6-96h as required.
8. Harvestcells and perform surface or intracellular staining as required.
(Refer to Protocol of Intracellular Staining Flow Cytometry for more details.)
9. Acquireby flow cytometry.

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

Protocol C provides a common procedure for activatingT cells prior to staining for intracellular cytokines, activation markers orcell proliferation using dendritic cells (DCs) that have been pulsed withantigen. Other types of antigen presenting cells may require different amountsof antigen and incubation times.

Reagents

• PBS
• Cell media
• Lipopolysaccharide (LPS)

Methods

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