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 method—no 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
Figure 2); 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 1—Three-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).
2
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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B
PBMC
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101
CD4 PerCP
C
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1.1%
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102
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CD4+CD25+
97%
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PE
CD25 PE
105
D
CD4+CD25–
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102
CD4 PerCP
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102
96%
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101
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102
103
CD4 PerCP
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102
103
104
105
PerCP
Figure 2—Presence 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
A
nanoparticles are left on the surface of the cells. The uptake of
100
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-
60
degradable nanoparticles.1,2
40
The Dynabeads® polymer coating prevents unwanted expo-
20
sure of your cells to iron oxides or dextran during isolation. Add-
0
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.
B
100
Mouse cells
CD4+CD25 +
Foxp3
Positive cells (%)
80
High Treg purity and functional capacity
CD25 and Foxp3 are expression markers associated with suppressive
60
n=9
n=6
function in Treg cells. A significantly higher number of cells express-
40
ing both the CD25 and Foxp3 markers is isolated using Dynabeads®
20
than using a column-based method (Figure 3).
The functionality of isolated CD4+CD25+ Treg cells can be exam-
0
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 3—Higher 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.
4
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 (Figure 4).
A
150
CD4+CD25–
125
54%
Count
100
75
50
25
0
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CFSE FITC
B
Expansion of isolated Treg cells
150
CD4+CD25–:CD4+CD25+
2%
Count
Because of the low proportion of Treg cells in blood (2–10%), it is
often necessary to expand isolated Treg cells to perform desired
experiments. The Dynabeads® CD3/CD28 T cell expansion tech-
50
25
nology mimics in vivo T cell activation via antigen-presenting cells
(APCs) and allows for gentle and efficient physiological ex vivo acti-
0
101
102
CFSE FITC
vation and expansion in both mouse and human settings.
You can obtain a 100-fold increase in the number of Treg
Figure 4—Suppressive 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
1
0.1
120
B
CD25+
CD25+ 2 stimulations
0
3
6
8
11
Time (days)
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16
21
103
100
80
60
40
20
0
0:1
1:16
1:8
1:2
1:1
Suppressor:responder ratio
Figure 5—Treg 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
research–grade 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 28:2572–2581.
2. Berry, C.C. et al. (2004) Biomaterials 25:5405–5413.
3. Valmori, D. et al. (2006) J Immunol 177:944–949.
of activation and expansion Dynabeads® includes a clinical
A
4. Battaglia, M. et al. (2006) J Immunol 177:8338–8347.
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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103
86%
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102
103
104
105
101
Foxp3 FITC
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105
Foxp3 FITC
CD25 and Foxp3 after expansion
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Dynabeads® method
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CD25 PE
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103
102
101
101
102
103
Foxp3 FITC
104
105
29%
103
102
101
Column-based method
104
54%
CD25 PE
B
102
101
102
103
104
105
Foxp3 FITC
Figure 6—Expanded 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.
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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 5–50 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.
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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