Download Low impact on cells, high impact on results

Positive Cell Isolation
Low impact on cells,
high impact on results
Dynabeads® FlowComp™ technology for positive
isolation of human and mouse cells
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Positive Cell Isolation
Low impact on cells, high impact on results
Dynabeads FlowComp™ technology
®
→→ Isolate from whole blood, buffy coat, or mononuclear cells
Isolate bead-free cells
→→ Release the cells and remove the beads
In contrast to column-based systems, the Dynabeads® FlowComp™
→→ Better purity and viability
technology allows you to isolate bead-free cells directly from human
→→ Tube-based method, no columns required
whole blood, buffy coat, or from mouse/human mononuclear cells
(MNC) (Figure 1). Following separation, the beads are immediately
The immune system protects us from harmful substances
released and removed, ensuring the least possible interaction with
through a complex interplay of immune cells. Cell isolation is the
your cells.
first of many steps in immunological workflows, and exposure of
your cells to certain foreign substances during this step can influ-
Column-free method
ence your results.
With the tube-based Dynabeads® technology, your cells are not
exposed to the stress of being passed through a dense column.
Avoid artifacts
Dynabeads® are larger and have a higher iron content, allowing
When using column-based systems with “biodegradable” nano-
for faster kinetics and better separation. The isolated bead-free
particles, the magnetic particles are left on the cells after positive
cells maintain their functional characteristics (Figure 2) and are
isolation. There is growing concern among scientists about what
ready for flow cytometric analysis or any downstream cell-based
the short- and long-term effects of the continued exposure of
assay [3–7]. Dynabeads® are very stable (shelf life >2 years), are not
cells to iron oxides, sugar (dextran), and other potentially harmful
biodegradable, and show no leakage of iron or other immuno-
substances may be [1–2].
gens that could potentially influence your results.
Dynabeads® bind to
target cells during
incubation.
Separate beads and cells
using the magnet.
Bead
Modified
streptavidin
Start with human whole
blood/buffy coat or MNC from
mouse/human samples. Incubate
with provided antibody mix and
add Dynabeads®.
Apply Dynal® magnet to
separate bead-bound cells.
Discard supernatant.
Add FlowComp™ buffer
to release target cells
from Dynabeads®.
Modified
biotin
Cell
Release
buffer
Isolated bead-free cells are
ready for use in any downstream application, including
flow cytometry.
Figure 1. Overview of the simple isolation procedure. The starting sample is mouse/human mononuclear cells (MNC) or human whole blood/buffy coat.
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Before isolation
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Propidium iodide
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Count
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Better purity and viability
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25
0
For easier flow cytometer read-out, some researchers choose to
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CD4+
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CD4+
only gate for the live cells when analyzing their cells. Thus, the
Column-based isolation of mouse CD4+ T cells
apoptotic or dead cells are excluded from the analysis, but not
Purity:
78.5%
400
from the sample itself. Dynabeads® FlowComp™ Mouse CD4 yield
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Propidium iodide
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cells), compared to as little as 49% recovery with a column-based
Count
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83% total recovery (total number of live, non-apoptotic CD4 +
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method (Figure 3). With Dynabeads® FlowComp™ technology,
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Viability:
63%
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0
cell purity is also consistently superior (Figure 4).
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10
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CD4+
CD4+
Dynabeads® FlowComp™ isolation of mouse CD4+ T cells
600
Purity:
97.2%
Freshly isolated Day 0
400
Count
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Propidium iodide
500
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300
CD14 APC-A
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0
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CD86 PE-A
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HLA DR FITC-A
Day 6
CD14 APC-A
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CD86 PE-A
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Viability:
86%
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CD4+
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Figure 3. Isolation of CD4+ T cells from mouse spleen cells. Cell isolation using
Dynabeads® FlowComp™ Mouse CD4 results in substantially higher purity
(97%) and viability (86%) than column-based positive cell isolation (yielding
purity and viability of 78% and 63%, respectively).
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CD14 APC-A
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CD4+
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HLA DR FITC-A
Figure 2. Isolated cells keep their functional characteristics. Dynabeads®
FlowComp™ Mouse CD14 was used to isolate monocytes from whole blood.
After 6 days of culturing with IL-4 and GM-CSF, the cells differentiated into
monocyte-derived dendritic cells (Mo-DC). As seen, CD14 was lost during culture and cells up-regulated HLA-DR and CD86. More than 60% recovery of
Mo-DC was observed. Similar results were observed when starting with MNC
or buffy coat.
Purity (%)
CD14 APC-A
200
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60
40
20
Dynabeads® FlowComp™
Human CD8 (n = 63)
Column-based
isolation (n = 5)
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Figure 4. Isolation of CD8+ T cells from peripheral blood mononuclear cells. Isolation
with Dynabeads® FlowComp™ Human CD8 gave high purity and minimal performance variations.
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www.invitrogen.com
”Love it; looks like over 90% recovery for the Dynabeads® FlowComp™ Mouse CD4
kit, even from raw splenocytes. I definitely intend to use it.” Bindu Kanathezhath, MD
Children’s Hospital and Research Center, Oakland, California USA
To learn more about the high recovery, viability, and purity you can get with Dynabeads® FlowComp™ technology,
visit www.invitrogen.com/flowcomp.
Ordering information
Product
Amount processed
Cat. No.
Dynabeads® FlowComp™ Human CD4
80 mL whole blood/buffy coat*/2 x 109 cells
113-61D
Dynabeads® FlowComp™ Human CD8
80 mL whole blood/buffy coat*/2 x 109 cells
113-62D
Dynabeads® FlowComp™ Human CD3
80 mL whole blood/buffy coat*/2 x 109 cells
Positive isolation of human cells
113-65D
9
Dynabeads® FlowComp™ Human CD14
80 mL whole blood/160 mL buffy coat/2 x 10 cells
113-67D
Dynabeads® FlowComp™ Human CD45RA
2 x 109 cells
113-68D
Dynabeads® FlowComp™ Mouse CD4
2 x 109 cells
114-61D
Dynabeads® FlowComp™ Mouse CD8
2 x 109 cells
114-62D
Dynabeads® FlowComp™ Mouse Pan T (CD90.2)
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2 x 10 cells
114-65D
Dynabeads® FlowComp™ Mouse CD4+CD25+ Treg Cells
1 x 109 cells
114-63D
Dynabeads® FlowComp™ Mouse CD49b
2 x 109 cells
114-64D
Dynabeads® FlowComp™ Flexi
2 x 109 cells
110-61D
DynaMag™ magnets
See www.invitrogen.com/magnets for magnet recommendations.
HulaMixer™ Sample Mixer
1 unit, holds 0.5 mL–50 mL tubes
Positive isolation of mouse cells
Related products
159-20D
* For isolation from buffy coat, contact Technical Support for specific protocols.
For current prices, please visit www.invitrogen.com.
Visit www.invitrogen.com/immunology for relevant antibodies, cell expansion technology, and flow cytometry reagents.
References
1. Pisanic TR II et al. (2007) Nanotoxicity of iron oxide nanoparticle internalization in growing neurons. Biomaterials 28:2572–2581.
2. Berry, CC et al. (2004) Cell response to dextran-derivatised iron oxide nanoparticles post internalisation. Biomaterials 25:5405–5413.
3. Feng X et al. (2010) Foxp1 is an essential transcriptional regulator for the generation of quiescent naive T cells during thymocyte development. Blood 115(3):510–518.
4. Del Cacho E et al. (2009) Avian follicular and interdigitating dendritic cells: Isolation and morphologic, phenotypic, and functional analyses. Vet Immunol Immunopathol 129:66–75.
5. Wang J et al. (2009) De novo generation and enhanced suppression of human CD4+CD25+ regulatory T cells by retinoic acid. J Immunol 183:4119–4126.
6. Brody JD et al. (2009) Immunotransplantation preferentially expands T-effector cells over T-regulatory cells and cures large lymphoma tumors. Blood 113(1):85–94.
7. Huber SA (2009) Depletion of γδ+ T cells increases CD4+ FoxP3 (T regulatory) cell response in coxsackievirus B3-induced myocarditis. Immunology 127:567–576.
DYNAL® has pioneered magnetic separation technologies for biological discovery that are 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
For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated.
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Invitrogen catalog or www.invitrogen.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. CO21311 0510