Activation and Expansion of Human Treg Cells
实验概要
This protocol is intended for activation and expansion of human Treg cells isolated with the Dynal® CD4 CD25 Treg Kit (Cat. no. 113.23D). The expanded Treg cells retain their regulatory capacity (1-3).
Dynabeads® Human Treg Expander are uniform 4.5 μm, superparamagnetic polystyrene beads coated with an optimized mixture of monoclonal antibodies against the CD3 and CD28 cell surface molecules of human T cells. The CD3 antibody coated on Dynabeads® Human Treg Expander is specific for the epsilon chain of human CD3, 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 coupled to the same bead, mimicking In vivo stimulation by antigen presenting cells.
实验原理
Dynabeads® Human Treg Expander offers a simple method for expansion of Treg cells that does not require antigen- presenting cells or antigen. Just add Dynabeads® Human Treg Expander and recombinant IL-2 (rIL-2) to the Treg culture to expand the Treg cells. Cell cultures showing signs of exhaustion can be re-stimulated by adding fresh beads and rIL-2.
Ready-to-use Dynabeads® Human Treg Expander offer the first artificial antigen- presenting cells to provide simultaneous signals to TCR/CD3 and CD28 for full activation and expansion of human Treg cells.
主要试剂
2 ml Dynabeads® Human Treg Expander
2 x 107 beads/ml in phosphate buffered saline (PBS), pH 7.4, w/ 0.1% human serum albumin (HSA).
Magnet: Any Dynal® MPC™
Buffer 1: PBS w/ 0.1% BSA, pH 7.4
Cultural Medium: OpTmizer™ T-Cell Expansion SFM (Gibco) serum free 1X formulation designed to support the culture and expansion of human T cells(or equivalent)
Round bottom tissue culture plates or tissue culture flasks of suitable size
Humidified CO2 incubator
Dynal® CD4 CD25 Treg Kit
rIL-2
实验步骤
1. Dynabeads® Washing Procedure
Dynabeads® should be washed before use with the aid of a magnet.
1) Resuspend the Dynabeads® in the vial.
2) Transfer the desired volume of Dynabeads® to a tube.
3) Add the same volume of Buffer 1 as the initial volume of Dynabeads®, or at least 1 ml, and mix.
4) Place the tube in a magnet for 1-2 minutes until the Dynabeads® are separated; discard the supernatant. Remove the tube from the magnet.
5) Resuspend the washed Dynabeads® in the same volume of Buffer 1 as the initial volume of Dynabeads®.
2. Isolation of Human Treg Cells
For isolation of human Treg cells it is recommended to use the Dynal® CD4 CD25 Treg Kit.
3. Expansion of Human Treg Cells Day 0:
Start with 1 x 105 Treg cells in 100 μl Culture Medium in a round bottom 96 well tissue culture plate.
Add 20 μl Dynabeads® Human Treg Expander.
Add 500 U/ml rIL-2.
Day 1:
Add 100 μl Culture Medium containing
500 U/ml rIL-2.
Day 3:
Resuspend and split the culture in half and add 100 μl Culture Medium containing
500 U/ml rIL-2 per well.
Day 5-7:
Split the culture when needed. Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures. Count the cells at least twice weekly after thorough resuspension.
When the cell density exceeds 2 x 106 cells/ml or when the medium becomes yellow, split cultures to a density of 0.5 - 1.0 x 106 cells/ml in Culture Medium containing 500 U/ml rIL-2. Grow the cells until the well is half-full (~500,000 cells) and transfer the cells from a 96 well to a 24 well plate.
Day 8:
Remove the Dynabeads® by resuspending the cells and transferring the cells to a suitable tube. Place the tube in a magnet for 1-2 minutes until the Dynabeads® are separated. Centrifuge the supernatant and resuspend the cell pellet in fresh Culture Medium containing 100 U/ml rIL-2. Split the cultures when needed and rest the cells until Day 21-24 in culture medium with 100 U/ml rIL-2. Incubate in a humidified CO2 incubator at 37°C.
4. Re-stimulation of Human Treg Cells
The cells can be re-stimulated 15-18 days after the first stimulation, or when cell shrinking and reduced rate of proliferation is observed.
1) Before re-stimulation:
i. Count the cells and split the cultures to a density of 1 x 106 cells/ml in Culture Medium.
ii. Use the volumes given in Table 2 and follow the protocol below.
2) Day 0:
i. Start with 1 x 106 Treg cells in 1 ml Culture Medium in a 24 well tissue culture plate.
ii. Add 50 μl Dynabeads® Treg Expander.
iii. Add 100 U/ml rIL-2.
3) Day 1:
i. Add 100 μl Culture Medium containing100 U/ml rIL-2 per well.
4) Day 2 or 3:
i. Resuspend and split the culture in half and add 100 μl Culture Medium containing 100 U/ml rIL-2 per well.
5) Day 5-7:
i. Split the culture when needed.
ii. Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures.
iii. Count the cells at least twice weekly after thorough resuspension.
When the cell density exceeds 2 x 106 cells/ml or when the medium becomes yellow, split cultures to a density of 0.5 - 1.0 x 106 cells/ml in Culture Medium containing 100 U/ml rIL-2.
6) Day 8:
i. Remove the Dynabeads® by resuspending the cells and transferring the cells to a suitable tube.
ii. Place the tube in a magnet for 1-2 minutes until the Dynabeads® are separated.
iii. Centrifuge the supernatant and resuspend the cell pellet in fresh Culture Medium containing 100 U/ml rIL-2.
iv. Split the cultures when needed and rest the cells until Day 21-24 in culture medium with 100 U/ml rIL-2.
注意事项
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.
This product contains 0.02% sodium azide as a preservative, which is cytotoxic. Avoid pipetting by mouth!
Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up.
