Physiological Activation of Human T Cells

This protocol is intended for physiological activation of human T cells, e.g. CD4+ T cells, CD8+ T cells, polyclonal T cells, or antigen specific T cells

Downstream Applications

The activated T cells can be analyzed shortly after activation (for transfection/ transduction or to study e.g. TCR signaling, proteomics or gene expression). T cells can be left in culture to differentiate into T helper cell subsets (ref 1,2,3), T cell proliferation or expansion of polyclonal/ Ag-specific T cells (ref 4,5).

Additional materials required

• Buffer: Phosphate buffered saline (e.g., Invitrogen Gibco Cat. No. 10010-023) with 0.1% bovine serum albumin and 2 mM EDTA, pH 7.4 (PBS w/0.1% BSA).

• Magnet (DynaMag™): See Magnets for Molecular and Cell Separation Applications for magnet recommendations.

• Culture medium: Advanced RPMI Medium 1640 (Invitrogen Gibco Cat. No. 12633-012) with 2 mM L-Glutamin, 10% FCS/FBS and 100 U/ml penicillin/streptomycin can be used. Alternatively CTS™ OpTmizer™ T Cell Expansion SFM (Invitrogen Gibco Cat. No. A1048501) with 100 U/ml penicillin/streptomycin, or an equivalent culture medium.

• Heat inactivated Fetal Calf Serum (FCS).
• Recombinant human IL-2.
• Flat bottom tissue culture plates or tissue culture flasks of suitable size.
• Humidified CO₂ incubator.

Critical notes

• Resuspend the Dynabeads™ in the vial carefully before use, i.e. vortex for >30 sec., or tilt and rotate for 5 minutes.
• This product should not be used with Dynal™ MPC™-1.
• Never use less than the recommended volume of Dynabeads™.
• Carefully follow the recommended pipetting volumes.
• Avoid air bubbles during pipetting.
• Prior to flow cytometric analysis, Dynabeads™ and bead-bound cells should be removed. Upon activation and for 2–3 days thereafter, some cells will bind strongly to the beads. Resuspend the bead/cell suspension thoroughly by pipetting to increase cell recovery, separate on a magnet (after transfer to a suitable tube) and collect supernatant containing the T cells. The bead-bound cell fraction can be cultured overnight and the above process repeated to further increase T cell recovery. When using cells for proteomics or gene expression studies, lyse the cells prior to bead removal.

This product allows for easy physiological activation of human T cells, without the need for preparing antigen-presenting cells (APCs) or antigen.

Preparations

• See Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species for recommended Dynabeads™ products for positive or negative isolation of all human T cells, or specific T cell subsets. Follow the procedure described in the respective package insert.
• Prepare cell culture medium.

Dynabeads™ Washing Procedure 

Dynabeads™ should be washed before use.

1. Resuspend the Dynabeads™ Human T-Activator CD3/CD28 in the vial.


2. Transfer the desired volume of Dynabeads™ to a tube.


3. Add an equal volume of Buffer, or at least 1 ml, and mix (vortex for 5 seconds, or keep on a roller for at least 5 min).


4. Place the tube on a magnet for 1 min and discard the supernatant.


5. Remove the tube from the magnet and resuspend the washed Dynabeads™ in the same volume of Culture Medium as the initial volume of Dynabeads™ taken from the vial (step 2).

Activation of Human T Cells

1. Start with 8 x 10⁴ purified T cells in 100–200 μl medium in a 96-well tissue culture plate.


2. Add 2 μl Dynabeads™ Human T-Activator CD3/CD28 to obtain a bead-to-cell ratio of 1:1 (see table 1).


3. Incubate in a humidified CO₂ incubator at 37°C, according to your specific experimental requirements.


4. Harvest the activated T cells and use directly for further analysis.


5. For flow cytometry applications, remove the beads prior to staining. Place the tube on a magnet for 1–2 minutes to separate the beads from the solution. Transfer the supernatant containing the cells to a new tube.

Expansion of Human T Cells

1. Start with 1–1.5 x 10⁶ purified T cells/ml in culture medium in a suitable tissue culture plate or tissue culture flask.


2. Add Dynabeads™ Human T-Activator CD3/CD28 at a bead-to-cell ratio of 1:1 (see table 1).


3. Add 30 U/ml rIL-2.


4. Incubate in a humidified CO₂ incubator at 37°C, according to your specific experimental requirements.


5. Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures.


6. Count the cells at least twice weekly after thorough re-suspension.


7. When the cell density exceeds 2.5 x 10⁶ cells/ml or when the medium turns yellow, split cultures back to a density of 0.5–1 x 10⁶ cells/ml in culture medium containing 30 U/ml rIL-2.

Re-Stimulation

Cell cultures showing signs of exhaustion (typically at day 7-10 of expansion) can be re-stimulated several times by adding fresh Dynabeads™ Human T-Activator CD3/CD28 and rIL-2. The CD8+ T cells remain cytotoxic after repeated re-stimulations. Re-stimulation is typically necessary when cell shrinking and a reduced rate of proliferation is observed. Guidelines for re-stimulation are provided in Table 2, although we recommend optimization for your particular application. Do not use an excess volume of Dynabeads™ Human T-Activator CD3/CD28, as excess Dynabeads™ per cell may inhibit expansion. Prior to re-stimulation, remove the used Dynabeads™ by transferring the cells to a suitable tube. Place the tube on a magnet for 1-2 minutes until the Dynabeads™ have moved to the side of the tube. Transfer the supernatant containing the cells to a new tube.

Continue as described below.

1. Count the cells and resuspend to a density of 1 x 10⁶ cells/ml in culture medium with 30 U/ml rIL-2 in a suitable culture plate or tissue culture flask.


2. Add Dynabeads™ Human T-Activator CD3/CD28 at a bead-to-cell ratio of 1:1.


3. Add 30 U/ml rIL-2.


4. Incubate in a humidified CO₂ incubator at 37°C for the length of your specific experiment.


5. Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures.


6. Count the cells at least twice weekly after thorough re-suspension.


7. When the cell density exceeds 2.5 x 10⁶ cells/ml or when the medium turns yellow, split cultures back to a density of 0.5-1 x 10⁶ cells/ml in culture.

Table 1: Volume recommendations for bead-to-cell ratio = 1:1 medium with 30 U/ml rIL-2.

Table 2:   Re-stimulation guidelines for anti-CD3/CD28–expanded cultures
 

* Establish optimal times for your particular cells. Please note that these are only generic guidelines

Manufactured by Invitrogen Dynal® AS. Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003. The latest revision of the package insert/ instruction for use is available on our website. For further technical information please visit our contact page.

Description of Materials

Dynabeads® Human T-Activator CD3/CD28 are uniform 4.5 μm, superparamagnetic polymer beads coated with an optimized mixture of monoclonal antibodies against the CD3 and CD28 cell surface molecules of human T cells. The CD3 antibody is specific for the epsilon chain of human CD3, which is considered to be a subunit of the TCR complex. The CD28 antibody is specific for the human CD28 co-stimulatory molecule, which is the receptor for CD80 (B7-1) and CD86 (B7-2). Both antibodies are mouse anti-human IgGs coupled to the same bead, mimicking in vivo stimulation by APCs. Both the bead size and the covalent antibody coupling technology are critical parameters to allow the simultaneous presentation of optimal stimulatory signals to the T cells in culture, thus allowing their full activation and expansion.

Related Dynabeads products

A comprehensive range of Dynabeads® for isolation of T cells and T cell subsets are available. Please visit the page Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species.

Storage and Stability

Store opened vials at 2-8°C and keep sterile. Do not freeze the product. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend the beads well before use. This product is stable until the expiration date stated on the label when stored unopened at 2-8°C.

Warnings & Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. Certificate of Analysis/Compliance is available upon request. Material Safety Data Sheet (MSDS) is available at .

1. Volpe E et al. (2008) A critical function for transforming growth factor-β, interleukin 23 and proinflammatory cytokines in driving and modulating human Th-17 responses. Nat. Immunol. 9(6):650-657.


2. De Fanis U et al. (2007) GATA3 upregulation associated with surface expression of CD294/CRTH2 : a unique feature of human Th cells. Blood 109(10):4343-4350.


3. Schade AE et al. (2008) Dasatinib, a small-molecule protein tyrosine kinase inhibitor, inhibits T-cell activation and proliferation. Blood 111(3):1366-1377.


4. Trickett A et al. (2003) T cell stimulation and expansion using anti-CD3/CD28 beads. J Imm Methods 275:251-255.


5. Ward FJ et al. (2008) Clonal regulatory T cells specific for a red blood cell autoantigen in human autoimmune hemolytic anemia. Blood 111(2):680-687.