Positive Cell Isolation

Pure and functional Treg cells

Isolate human and mouse regulatory T cells with Dynabeads

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Positive Cell Isolation

Human and mouse regulatory CD4+CD25+ T cells

Dynabeads for gentle cell isolation

Isolate functional CD4+CD25+ Treg cells with high expression of Foxp3

Tube-based methodno columns needed
No beads remain bound to the cell surface
Isolated Treg cells can be expanded and retain their suppressive capacity and Treg phenotype

Two kits, the Dynabeads Regulatory CD4+CD25+ T Cell Kit and

The combination of negative isolation of CD4 + cells followed

Dynabeads FlowComp Mouse CD4+CD25+ Treg Cells, offer an

by positive isolation of CD25+ cells secures high purity, yield, and

easy-to-use, flow-compatible method for positive isolation of

viability of the small Treg population. You can isolate highly func-

regulatory T cells (Treg cells). A simple three-step isolation pro-

tional Treg cells with up to 97% purity (CD4+CD25+ expression,

cedure (Figure 1) leaves cells bead-free. The isolated cells can be

Figure2); on average, more than 80% of the isolated CD25+ cells

directly analyzed by flow cytometry or used in any downstream

express the transcription factor Foxp3.

experiment.

Start with PBMC or mouse

leukocytes. Add Antibody Mix
and Depletion Dynabeads for
removal of non-CD4+ cells

Add Dynabeads CD25 for

positive isolation of CD25+ cells

Remove
CD25 cells

Transfer
negatively
isolated
CD4+ cells
CD4+
CD25

CD4+
CD25+

Add release agent for

release of beads from the
positively isolated Treg cells

CD4+
CD25
CD4+
CD25+
CD4+
CD25+

Transfer the isolated

bead-free Treg cells to a
new tube; cells are ready
for analysis

Use a
magnet to
separate
beads and
cells
CD4+
CD25+

CD4+
CD25+

CD4+
CD25+

CD4+
CD25+

CD4+
CD25+

STEP 1

STEP 2

STEP 3

Figure 1Three-step procedure for isolation of Treg cells. Step 1, untouched CD4 + T cells are negatively isolated using Depletion Dynabeads. Step 2, CD25+ cells are
positively isolated using Dynabeads CD25. Step 3, beads are released from the CD4 +CD25+ cells using the provided release reagent (FlowComp release buffer
for the mouse product, DETACHaBEAD release reagent for the human product. Note that DETACHaBEAD reagent also releases the CD25 antibody from the cell
surface).

Regulatory T cells
Regulatory CD4+CD25+ T cells are a specialized low-abundance sub-

accelerated by evidence from experimental mouse and human

population of T cells that act to maintain homeostasis within the

models demonstrating that the immunosuppressive potential of

immune system. Recently, their critical role in regulating the immune

these cells can be utilized in the treatment of various conditions such

response has been firmly established. Interest in Treg cells has been

as autoimmune diseases, infectious diseases, and cancer.

A

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2.5%

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7%

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CD4+

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CD25 PE

CD25 PE

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PBMC

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CD4 PerCP

C

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1.1%

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CD4+CD25+

97%

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PE

CD25 PE

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CD4+CD25

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CD4 PerCP

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96%

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CD4 PerCP

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PerCP

Figure 2Presence and percentage of human CD4+CD25+ cells during the three-step isolation process using the Dynabeads Regulatory CD4+CD25+ T Cell Kit. A. In the PBMC
sample, 2.5% of the cells were CD4 +CD25+. B. After negative isolation of CD4 + cells, 7% of the CD4 + cells were CD25+. C. After isolation of CD25+ cells using Dynabeads
CD25, ~1% of the low-expressing CD25+ cells remained in the CD4 +CD25 fraction. D. The isolated CD4 +CD25+ cells were 97% pure. Similar results are seen for mouse
Treg cells isolated using the Dynabeads Mouse Regulatory CD4 +CD25+ T Cell Kit (not shown).

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Positive Cell Isolation

Unaffected integrity of isolated Treg cells

A unique advantage of the Dynabeads technology is the ability to isolate bead-free Treg cells. This is in contrast to alternative commercially available isolation methods in which magnetic
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nanoparticles are left on the surface of the cells. The uptake of

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Human cells
CD4+CD25+
Foxp3

Positive cells (%)

80

such particles may be related to cytotoxic/immunogenic effects

on cells, a problem recently recognized when working with bio-

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degradable nanoparticles.1,2

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The Dynabeads polymer coating prevents unwanted expo-

20

sure of your cells to iron oxides or dextran during isolation. Add-

ing a release reagent separates the cells and the Dynabeads in

Dynabeads
isolation

Column-based
isolation

one simple step. The integrity of your Treg cell population is not
impacted by the gentle, tube-based isolation method.

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Mouse cells
CD4+CD25 +
Foxp3

Positive cells (%)

80

High Treg purity and functional capacity

CD25 and Foxp3 are expression markers associated with suppressive

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n=9

n=6

function in Treg cells. A significantly higher number of cells express-

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ing both the CD25 and Foxp3 markers is isolated using Dynabeads

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than using a column-based method (Figure 3).

The functionality of isolated CD4+CD25+ Treg cells can be exam-

Dynabeads
isolation

Column-based
isolation

ined by co-culturing them with CD4+CD25 effector T cells. As an

example, the Dynabeads Regulatory CD4+CD25+ T Cell Kit was used

Figure 3Higher numbers of human and mouse CD4+CD25+ Foxp3+ T cells after isolation using Dynabeads. A. Human Treg cells were isolated from two different
donors using the Dynabeads Regulatory CD4+CD25+ T Cell Kit and a columnbased method. B. Mouse Treg cells were isolated from mouse spleen cells using
Dynabeads FlowComp Mouse CD4 +CD25+ Treg Cells and a column-based
method. Treg purity was identified by flow cytometric analysis of CD25 and
Foxp3.

to isolate both the CD4+CD25+ and CD4+CD25 human cell populations. In a suppression assay that followed, the isolated Treg cells
profoundly inhibited the proliferation of CFSE-labeled CD4+CD25
T cells (Figure4).

A

150
CD4+CD25
125

54%
Count

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75
50
25
0

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CFSE FITC

Expansion of isolated Treg cells

150

CD4+CD25:CD4+CD25+

2%
Count

Because of the low proportion of Treg cells in blood (210%), it is

often necessary to expand isolated Treg cells to perform desired
experiments. The Dynabeads CD3/CD28 T cell expansion tech-

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25

nology mimics in vivo T cell activation via antigen-presenting cells

(APCs) and allows for gentle and efficient physiological ex vivo acti-

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CFSE FITC

vation and expansion in both mouse and human settings.

You can obtain a 100-fold increase in the number of Treg

Figure 4Suppressive capacity of isolated human Treg cells. A. Human CD4 +CD25
cells were CFSE-stained and stimulated with Dynabeads CD3 (bead:cell ratio
1:1). On day 4, 54% of the cells were dividing as identified by flow cytometry. B. Human CD4 +CD25 cells were CFSE-stained and stimulated with
Dynabeads CD3 in the presence of human CD4 +CD25+ Treg cells in a 1:1 ratio.
After 4 days, only 2% of the CD4 +CD25 cells were dividing; 96% suppression
of cell division was achieved in the presence of the human CD4 +CD25+ Treg
cells (left peak in histogram shows human Treg cells not CFSE-stained). Similar
results are seen with mouse Treg cells isolated using the Dynabeads Mouse
Regulatory CD4 +CD25+ T Cell Kit (not shown).

cells (Figure 5A), and the specific stimulation and expansion may
be enhanced using rapamycin.3,4 Using Dynabeads for isolation
and expansion, you can always count on good yields of highly
pure Treg cells, and avoid having to perform multiple isolations
using less efficient techniques.

A

10,000

Proliferation of CD25
responder cells (%)

Fold expansion

1,000

100

10

0.1

120

CD25+
CD25+ 2 stimulations

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Time (days)

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16

21

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100
80
60
40
20
0

0:1

1:16

1:8

1:2

1:1

Suppressor:responder ratio

Figure 5Treg cells activated with Dynabeads CD3/CD28 T cell expansion technology can be expanded 100-fold while retaining their Treg phenotype and functionality. A. A
higher expansion number can be obtained through restimulation at day 8 with >97% pure CD4 +CD25+ Treg cells to avoid overgrowth of non-Treg cells. B. Expanded
human Treg cells were co-cultured with allogenic PBMC for 6 days at various suppressor:responder ratios. Proliferation was measured using a standard thymidine
incorporation assay. At a 1:1 ratio, 80% suppression was achieved.

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Positive Cell Isolation

Treg suppressive capacity is maintained

The expanded Treg cells are functional and can suppress effector

researchgrade product, allowing you to move from mouse

T cells in a mixed lymphocyte culture (MLC) reaction (Figure 5B).

studies to clinical research (Phase l/ll) using the same technol-

The Treg cells retain their Foxp3 expression, and a higher purity is

ogy platform.

seen after both isolation and expansion using Dynabeads than is

seen with a column-based method (Figure 6).
Expanded mouse T cells can be used for further in vitro
manipulations, or for adoptive transfer in vivo. Our full portfolio

References
1. Pisanic, T.R., II et al. (2007) Biomaterials 28:25722581.
2. Berry, C.C. et al. (2004) Biomaterials 25:54055413.
3. Valmori, D. et al. (2006) J Immunol 177:944949.

of activation and expansion Dynabeads includes a clinical

4. Battaglia, M. et al. (2006) J Immunol 177:83388347.

CD25 and Foxp3 before expansion

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Dynabeads method

105 Column-based method

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90%

CD25 PE

CD25 PE

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86%
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Foxp3 FITC

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Foxp3 FITC

CD25 and Foxp3 after expansion

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Dynabeads method

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CD25 PE

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Foxp3 FITC

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29%

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Column-based method

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54%

CD25 PE

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Foxp3 FITC

Figure 6Expanded mouse Treg cells retain Foxp3 expression. The purity of mouse Treg cells isolated with the Dynabeads FlowComp Mouse CD4 +CD25+ Treg Cells
kit and a column-based method, before (A) and after (B) 10 days of expansion with Dynabeads Mouse CD3/CD28 T Cell Expander, as measured by CD25 and Foxp3
expression.

Ordering information
Quantity

Cat. no.

Product

Application

Dynabeads Regulatory CD4+CD25+ T Cell Kit

The kit contains Antibody Mix, Depletion Dynabeads, Dynabeads CD25,
and DETACHaBEAD release reagent

Human Treg cell isolation

Processes 1 x 109 cells

113-63D

Dynabeads FlowComp Mouse CD4+CD25+ Treg Cells

The kit contains Antibody Mix, Depletion Dynabeads, FlowComp Dynabeads (mTreg),
FlowComp Mouse CD25 Antibody, and FlowComp Release Buffer

Mouse Treg cell isolation

Processes 1 x 109 cells

114-63D

Magnets
DynaMag-15 (holds 4 standard 15 ml tubes, alternatively 4 x 5 ml tubes used in flow cytometry)

Magnetic separation

1 unit

123-01D

DynaMag-50 (holds 2 x 550 ml tubes)

Magnetic separation

1 unit

123-02D

See www.invitrogen.com/magnets for other magnet recommendations.

Related human products

Dynabeads Untouched Human CD4 T Cells

Cell isolation

Processes 1 x 109 cells

113-46D

Dynabeads CD3

Cell isolation

5 ml

111-51D

Dynabeads CD25

Cell isolation

5 ml

111-57D

Dynabeads CD3/CD28 T Cell Expander

T cell expansion

2 ml

111-31D

Dynabeads CD3/CD28 T Cell Expander

T cell expansion

10 ml

111-32D

Dynabeads Human Treg Expander

Treg cell expansion

2 ml

111-29D

Dynabeads CD3/CD28

Preclinical cell expansion

10 ml

111-41D

Dynabeads ClinExVivo CD3/CD28

Clinical-grade cell expansion

10 ml

402-03D

Processes 1 x 109 cells

114-15D

Related mouse products

Dynal Mouse CD4 Cell Negative Isolation Kit

Cell isolation

Dynabeads Mouse CD3/CD28 T Cell Expander

T cell expansion

2 ml

114-52D

Dynabeads Mouse CD3/CD28 T Cell Expander

T cell expansion

10 ml

114-53D

For current prices, please visit www.invitrogen.com.

Learn more about the best starting point for your T cell research at www.invitrogen.com/cellisolation.
Did you know that Dynabeads magnetic separation technology was pioneered in the 1980s by the Norwegian
company Dynal, now part of Invitrogen? To learn more, visit www.invitrogen.com/dynal.

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DYNAL has pioneered magnetic separation technology for biological discovery that is both simple and highly reproducible. Based
on their patented superparamagnetic, monodisperse beads, Dynabeads technologies represent a superior paradigm for cell and
biomolecule separation in a wide range of basic and clinical research applications, diagnostic assays, and therapeutic protocols.

www.invitrogen.com
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terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated. B-079497-r1 1008