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Introduction

Product Contents

Dynabeads® Mouse T-Activator CD3/CD28

1 × 0.4 ml (Cat. no. 114.56D)
1 × 2 ml (Cat. no. 114.52D)
5 × 2 ml (Cat. no. 114.53D)

Note that Cat. no. 114.52D and 114.53D are formerly know as Dynabeads® Mouse 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 mouse T cells, e.g. CD4+ T cells, CD8+ T cells, T regulatory cells (Treg), 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 www.lifetechnologies.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, or an equivalent culture medium.
  • Heat inactivated Fetal Calf Serum (FCS). Recombinant mouse IL-2 (human IL-2 can also be used).
  • 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.
  • For research use only.

Protocol

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

Preparations

  • See www.lifetechnologies.com/cellisolation for recommended Dynabeads® products  for positive or negative isolation of all mouse T cells, or specific T cell subsets.  Follow the procedure described in the respective package insert.
  • Note that for isolation of Treg cells (flow sorting or magnetic bead isolation),  it is critical to use an anti-CD25 antibody that do not block the binding of IL-2  if cells are used for expansion. Dynabeads® FlowComp™ Mouse CD4+CD25+  Treg Cells (Cat. no. 114.63D) can be used.
  • Prepare cell culture medium.


Dynabeads Washing Procedure

Dynabeads should be washed before use.

  1. Resuspend the Dynabeads Mouse 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 Mouse 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 Mouse 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. 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.

  5. Transfer the supernatant containing the cells to a new tube.



Expansion of Mouse 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 Mouse 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 × 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 Mouse 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 Mouse 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.

  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 suitable culture plate or tissue culture flask.

  2. Add Dynabeads Mouse 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.


Expansion of Mouse Regulatory T Cells

  1. Start with 1-1.5 × 106 cells/ml culture medium in a suitable tissue culture plate  (105 total cells per well in a 96-well plate, 1-1.5 × 106 total cells per well in a  24 well plate).

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

  3. Add 2,000 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  containing 2,000 U/ml rIL-2.


Treg cells retain FoxP3 expression after 2 weeks expansion.

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

8 × 104 T cells 1 × 106 T cells 35 × 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® Mouse T-Activator CD3/CD28 2 μl 25 μl 875 μl
rIL-2 30 U/ml 30 U/ml 30 U/ml
Seeding volume (medium) 100-200 μl 1-2 ml 35-70 ml


* Average number of T cells obtained from one mouse spleen.

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.

References

  1. McGeachy MJ et al. (2007) TGF-beta and  IL-6 drive the production of IL-17 and IL-10  by T cells and restrain Th-17 cell-mediated  pathology. Nat. Immunol. 8(12):13901397.

  2. Kimura A et al. (2007) IL-6-dependent and – independent pathways in the development  of interleukin 17-producing T helper cells.  PNAS 104(29):12099-12104.

  3. Izcue  A et al. (2008) Interleukin-23  restrains regulatory T cell activity to drive T  cell-dependent colitis. Immunity 28(4):559570.

  4. Li J et al. (2007) Transfer of in vitro expanded  T lymphocytes after activation with dendritomas  prolonged survival of mice challenged  with EL4 tumor cells. Int. J Oncol.  31(1):193-197.

  5. Masteller  EL et al. (2005) Expansion of  functional endogenous antigen-specific  CD4+CD25+ regulatory T cells from non-obese diabetic mice. J Immunol. 175: 30533059.
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114.52D_53D_56D.indd     Rev 003     5-May-2009