Protocols

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Activation of Immune Cells for Cytokine Production

I. Introduction

Immune cells can be stimulated by various means to produce specific cytokines. Here we provide some common methods for the induction of cytokines for human, mouse, and rat cells. Secreted cytokines can be quantitatively measured by ELISA (for more information visit: http://www.bdbiosciences.com/features/products/display_product.php?keyID=70) or Cytometric Bead Array (CBA) (for more information visit: http://www.bdbiosciences.com/pharmingen/cba). The frequencies of cytokine-producing cells can be measured by enzyme-linked immunospot (ELISPOT) (for more information visit: http://www.bdbiosciences.com/pharmingen/elispot) or by flow cytometric analysis of intracellular cytokines. The examination of intracellular cytokines is optimized by the use of protein transport inhibitors such as Brefeldin A (BD GolgiPlug, cat. no. 555029) or Monensin (BD GolgiStop, cat. no. 554724) that prevent the secretion of cytokines, thus trapping them in intracellular compartments. For a complete protocol please view our protocol titled "Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis".

II. Activation of Cells

A. Cultures for Generating Human Cytokine-Producing Cells

1. IL-3⁺ , IL-4⁺ , IL-5⁺ , IL-13⁺ and GM-CSF⁺ human cells: Human PBMC, purified human CD4⁺ or CD8⁺ cells (especially for IL-5⁺ and IL-13⁺ cells) are stimulated with immobilized anti-human CD3 antibody (clone UCHT1, 10 µg/ml for plate coating, cat. no. 555329), soluble anti-human CD28 antibody (clone CD28.2, 2 µg/ml; cat. no. 555725), recombinant human IL-2 (10 ng/ml; cat. no. 554603) and recombinant human IL-4 (20 ng/ml; cat. no. 554605) for 2 days. The cells are washed and subsequently cultured in medium containing rhIL-2 and rhIL-4 for another 3 days. Finally, the cells are harvested and restimulated for 4 hr with PMA (5 ng/ml; Sigma, cat. no. P-8139), calcium ionophore A23187 (250 ng/ml; Sigma, cat. no. C-9275), or ionomycin (500 ng/ml; Sigma, cat. no. I-0634) in the presence of a protein transport inhibitor (either GolgiStop, cat. no. 554724 or GolgiPlug, cat. no. 555029) if intracellular staining is desired.
2. TNF- α⁺ human cells: Human PBMC are stimulated with immobilized anti-human CD3 antibody (clone UCHT1, 10 µg/ml for plate coating, cat. no. 555329) and recombinant human IL-2 (10 ng/ml; cat. no. 554603) for 2 days. The cells are washed and subsequently cultured in medium containing rhIL-2 for another 3 days. Finally, the cells are harvested and restimulated for 4 hr with PMA (5 ng/ml; Sigma, cat. no. P-8139), calcium ionophore A23187 (250 ng/ml; Sigma, cat. no. C-9275), or ionomycin (500 ng/ml; Sigma cat. no. I-0634) or alternatively the cells can be restimulated with anti-CD3 and anti-CD28. Restimulation should be performed in the presence of a protein transport inhibitor (either GolgiStop, cat. no. 554724 or GolgiPlug, cat. no. 555029) if intracellular staining is desired.
3. IL-2+ , TNF- α⁺ , and IFN- γ +⁺ human cells: Human PBMC are stimulated for 6 hr with PMA (5 ng/ml; Sigma, cat. no. P-8139), calcium ionophore A23187 (500 ng/ml; Sigma, cat. no. C-9275), or ionomycin (500 ng/ml; Sigma cat. no. I-0634) in the presence of a protein transport inhibitor (either GolgiStop, cat. no. 554724 or GolgiPlug, cat. no. 555029) if intracellular staining is desired.
4. IL-1α ⁺ , IL-6⁺ , IL-8⁺ and GRO- α⁺ human cells: Human PBMC are stimulated for 4 hr with LPS (1.0 µg/ml; Sigma cat. no. L-8274) in the presence of a protein transport inhibitor (either GolgiStop, cat. no. 554724 or GolgiPlug, cat. no. 555029) if intracellular staining is desired.
5. IL-10⁺ , MCP-1⁺ , MIP-1 α⁺ , MCP-3⁺ , and MIG⁺ human cells: Human PBMC are stimulated for 24 hr with LPS (1.0 µg/ml) in the presence of a protein transport inhibitor if intracellular staining is desired.
6. IL-12⁺ human cells: Human PBMC are primed for 2 hr with rhIFN- γ (10 ng/ml; cat. no. 554616) and are then activated with IFN- γ (10 ng/ml) and LPS (1.0 µg/ml; Sigma, cat. no. L-8274) in the presence of a protein transport inhibitor (either GolgiStop, cat. no. 554724 or GolgiPlug, cat. no. 555029) for an additional 22 hr if intracellular staining is desired.
7. RANTES⁺ human cells: Because T cells can make RANTES constitutively (although its expression is upregulated by cell stimulation), human PBMC can simply be cultured for 24 hr in the presence of a protein transport inhibitor (GolgiStop™ is preferred) if intracellular staining is desired.

B. Human T cell Activation. Clone HIT3a (cat. no. 555336) is optimal for use in soluble format at 1 µg/ml, whereas clone UCHT1 (cat. no. 555329) is optimal in immobilized format (antibody coated onto plate at 5-10 µg/ml). The following suggested protocol for functional studies is from the datasheet for the anti-human CD28 antibody (cat. no. 555725): http://www.bdbiosciences.com/external_files/pm/doc/tds/human/live/web_enabled/33740D_555725.pdf

1. Isolate human PBMC with Ficoll-Paque^TM Plus.
2. Suspend PBMC in medium supplemented with 1% glutamine, 1% penicillin/streptomycin and 10% FBS at 10⁶/ml.
3. Incubate 1 x 10⁶ PBMC with soluble NA/LE format of CD3 mAb (cat. no. 555336) at 1 µg/ml final concentration in culture for 3 days. Alternatively, cells can be added to CD3 mAb (cat. no. 555329)-coated plates (5-10 µg/ml).
4. Wash cells twice and resuspend in the above medium at 10⁶/ml.
5. Distribute cells in a 96-well round bottom plate at 10⁵/well.
6. Add soluble NA/LE format of CD28.2 mAb at 5 µg/ml final concentration.
7. Incubate the plate for an additional 3 days.

C. Cultures for Generating Mouse Cytokine-Producing Cells

1. IL-2⁺ , TNF- α⁺ , and IFN- γ⁺ mouse cells: Mouse splenocytes are stimulated for 4 hr with PMA (5 ng/ml; Sigma, cat. no. P-8139) and ionomycin (500 ng/ml; Sigma, cat. no. I-0634) in the presence of a protein transport inhibitor (either GolgiStop, cat. no. 554724 or GolgiPlug, cat. no. 555029) if intracellular staining is desired.
2. IL-3⁺ , IL-4⁺ , IL-5⁺ , IL-10⁺ , GM-CSF⁺ mouse cells: Purified CD4⁺ mouse splenocytes from 6-month old BALB/c mice are stimulated with plate-bound anti-mouse CD3 (clone 145-2C11, 25 µg/ml; cat. no. 553057) and soluble anti-mouse CD28 (clone 37.51, 2 µg/ml; cat. no. 553294) for 2 days in culture together with rmIL-2 (10 ng/ml; cat. no. 550069) and rmIL-4 (50 ng/ml; cat. no. 550067), followed by a 3-day incubation with only rmIL-2 and rmIL-4. This is followed by a 4-hr stimulation with plate-bound anti-mouse CD3 (25 µg/ml) and anti-mouse CD28 (2 µg/ml) in the presence of a protein transport inhibitor if intracellular staining is desired. Alternatively, can restimulate with PMA (5 ng/ml; Sigma, cat. no. P-8139) and ionomycin (500 ng/ml; Sigma, cat. no. I-0634) with or without protein transport inhibitor (either GolgiStop, cat. no. 554724 or GolgiPlug, cat. no. 555029).
3. IL-6⁺ , IL-12⁺ , TNF- α⁺ mouse cells: 3-day thioglycolate elicited peritoneal cells are harvested and stimulated with 1 µg/ml LPS and GolgiPlug™ for 4 hr.
4. MCP-1⁺ mouse cells: Thioglycolate-elicited peritoneal macrophages from 6-month old BALB/c mice are stimulated with LPS (1 µg/ml; Sigma, cat. no. L-8274) overnight with or without a protein transport inhibitor (either GolgiStop, cat. no. 554724 or GolgiPlug, cat. no. 555029).

D. Mouse T cell Activation. Protocol for CD3 (clone 145-2C11, cat. no. 553057) plate stimulation of murine T cells for the detection of activation markers.

1. Coat a dish, flask, or plate with 10 µg/ml anti-mouse CD3 in sterile PBS overnight at 4°C.
2. The next day, wash 3 times with 10 ml sterile PBS.
3. Add 2 x 10⁶ splenocytes/ml and incubate up to 24-96 hours at 37°C. A precise time should be determined by performing a timecourse experiment and assay for your activation marker.

E. Cultures for Generating Rat Cytokine-Producing Cells

1. IL-4⁺ and IL-10⁺ rat cells: Purified splenic CD4⁺ cells from an adult rat are stimulated with plate-bound anti-rat CD3 (clone G4.18, 25µg/ml; cat. no. 554829) and soluble anti-rat CD28 (clone JJ319, 2 µg/ml; cat. no. 554993) for 2 days in culture together with recombinant rat IL-2 (10 ng/ml; cat. no. 555106) and rrIL-4 (50 ng/ml; cat. no. 555107), followed by a 3-day incubation with only rrIL-2 and rrIL-4. This is followed by a 4-6 hr stimulation with PMA (5 ng/ml; Sigma, cat. no. P-8139) and ionomycin (500 ng/ml; Sigma, cat. no. I-0634) in the presence of a protein transport inhibitor (either GolgiStop, cat. no. 554724 or GolgiPlug, cat. no. 555029). Alternatively, one can restimulate with plate-bound anti-rat CD3 and soluble anti-rat CD28 for 4-6 hr in the presence of a protein transport inhibitor if intracellular staining is desired.