Dynabeads® Human T-Activator CD3/CD28 - for physiological activation of human T cells

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Introduction

Product Contents

Dynabeads® Human T-Activator CD3/CD28

1 × 0.4 ml (Cat. no. 111.61D)
1 × 2 ml (Cat. no. 111.31D)
5 × 2 ml (Cat. no. 111.32D)
Note that Cat. no. 111.31D and 111.32D are formerly know as Dynabeads® CD3/CD28 T Cell Expander. Each product contains 4 × 107 beads/ml in phosphate buffered saline (PBS), pH 7.4, with 0.1% human serum albumin (HSA)

Product Description

This product is intended for physiological activation of human T cells, e.g. CD4+ T cells, CD8+ T cells, polyclonal   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 www.invitrogen.com/magnets 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 OpTmizer™ T Cell Expansion SFM (Invitrogen Gibco Cat. no. 0080022SA) 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.
 
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Protocols

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

Preparations

  • See www.invitrogen.com/cellisolation for recommended Dynabeads®  products for positive or negative isolation of all human T cells, or specific T cell subsets.
  • 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 × 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.

  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 × 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.

  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 × 106 cells/ml or when the medium turns yellow, split cultures back to a density of 0.5-1 × 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. 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 × 106 cells/ml in culture medium with 30 U/ml rIL-2 in a suitabl  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 × 106 cells/ml or when the medium turns yellow, split cultures back to a density of 0.5-1 × 106 cells/ml in culture medium with 30 U/ml rIL-2.

Table 1: Volume recommendations for bead-to-cell ratio = 1:1

  8 × 104 T cells 1 × 106 T cells 50 × 106 T cells
Type of culture plate/flask Per well in 96-well plate Per well in 24-well plate 175 cm2 tissue culture flask
Dynabeads® Human T-Activator CD3/CD28 2 μl 25 μl 1,250 μl
rIL-2 30 U/ml 30 U/ml 30 U/ml
Seeding volume (medium) 100-200 μl 1-2 ml 50-100 ml


Table 2: Re-stimulation guidelines for anti-CD3/CD28–expanded cultures
 
Cell type First re-stimulation* Subsequent re-stimulations*
CD4+ (polyclonal) 8-10 days 8-11 day intervals
CD8+ (polyclonal) 7-9 days 7-10 day intervals
T cells 7-9 days 10-12 day intervals

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

General Information

Certification

Invitrogen Dynal® AS conforms to the Quality Systems Standard ISO 9001:2000 and ISO 13485:2003 with the following scope: "Development, manufacturing, marketing and sales of Dynospheres®, Dynabeads® and associated products to customers that work within immunology, biological and clinical research, cell based therapy and in vitro diagnostics." In the United States, Dynabeads® ClinExVivo™ CD3/CD28 is available for use in clinical trials under an approved IND or IDE.

USA (Device Master File)

A Device Master File is held with the United States Food & Drug Administration (FDA), which will assist users with their application for FDA approvals on their clinical trials. If cross-referencing the Device Master File is of interest to an Investigational New Drug (IND) Application or other applications, please contact Invitrogen Dynal® with the sponsor’s and/or investigator’s full name and address, along with project name and aim. This information is required by Invitrogen Dynal® to issue a Letter of Authorisation, informing the FDA who has been authorised to cross-reference the Master File for their IND application.

Technical Service

Please contact Invitrogen Dynal® AS for further technical information at www.invitrogen.com Certificate of Analysis (CoA) is available upon request.

Precautions

Material Safety Data Sheet (MSDS) is available at http://www.invitrogen.com.

References

  1. Volpe E et al. (2008) A critical function for transfoming 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.
111.31D_32D_61D.indd   Rev 005    5-May-2009
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