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Dynabeads® Mouse T-Activator CD3/CD28 – for physiological activation of mouse T cells

互联网2013-11-13

1561

实验试剂

Buffer: Phosphate buffered saline 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 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).

实验设备

Flat bottom tissue culture plates or tissue culture flasks of suitable size.

Humidified CO₂ incubator.

实验步骤

1. 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).

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.

3. 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.

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.

4. 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.

5. 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.

8) Treg cells retain FoxP3 expression after 2 weeks expansion.

注意事项

1. Resuspend the Dynabeads in the vial carefully before use, i.e. vortex for >30 sec., or tilt and rotate for 5 minutes.

2. This product should not be used with Dynal MPC™-1.

3. Never use less than the recommended volume of Dynabeads.

4. Carefully follow the recommended pipetting volumes.

5. Avoid air bubbles during pipetting.

6. 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.

7. For research use only.

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