Cat. no. 110.60D
Rev. no. 001
000
Dynabeads® FlowComp™ Flexi, part A
– flow compatible and tube-based cell isolation
For research use only
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5.
KIT CONTENTS
PRODUCT DESCRIPTION
PROTOCOL
GENERAL INFORMATION
TECHNICAL RECOMMENDATIONS
1. KIT CONTENTS
Dynabeads FlowComp Flexi
Catalog no.
110.61D
Number of cells processed: up to 2x109
This kit includes:
Part A - cat. no. 110.60D:
Dynabeads FlowComp Flexi, part A
Content:
FlowComp Dynabeads (15 mg/ml)* 3 ml
FlowComp Release Buffer
2 x 20 ml
Part B - cat. no. D-20655:
DSB-X™ Biotin Protein Labeling Kit
Content:
See separate package insert
Part A and B are supplied in separate
boxes.
* 15 mg/ml equals approx. 1x109 beads/ml
in phosphate buffered saline (PBS), pH 7.4,
containing 0.1 % bovine serum albumin
(BSA) and 0.02 % sodium azide (NaN3).
Label your antibodies
using the supplied kit.
Incubate your single cell
s upsension with the
labeled antibodies.
Add FlowComp
Dynabeads. The
Dynabeads bind to the
target cells during
incubation.
Apply the magnet to
separate bead-bound
cells . Discard the
supernatant.
Remove the tube from the
magnet and add
FlowComp Releas e
Buffer to detach the
Dynabeads from the cells.
Remove the beads using
the magnet and transfer
the supernatant to a new
tube.
Isolated cells maybe used
in any downstream
application including flow
cytometry.
Fig. 1: The Dynabeads FlowComp Flexi workflow.
110.60D.indd 1
2. PRODUCT DESCRIPTION
This product is intended for positive isolation
of cells from a variety of samples and species,
e.g MNCs, tissue digests and splenocytes/
lymph node cells.
Principle of isolation
1. Label your antibody of choice using the
supplied DSB-X Biotin Protein Labeling Kit
2. Incuabate your cells with the DSB-X labeled
antibody
3. Add FlowComp Dynabeads to the labeled
cells, and isolate the bead-bound cells
using a magnet
4. Release target cells from Dynabeads using
the FlowComp Release Buffer.
Downstream applications
Isolated cells are bead-free and may be
used in any downstream application including flow cytometry. For recommended products and protocols visit www.invitrogen.com/
immunology.
Additional materials required
• Isolation Buffer: Ca2+ and Mg2+ free phosphate buffered saline (PBS) (e.g. Gibco
cat. no. 14190-094) supplemented with
0.1 % BSA and 2mM EDTA.
• Mixer allowing both tilting and rotation.
• Magnet: We recommend DynaMag-15 (cat.
no. 123.01D) for 1-15 ml samples and
DynaMag-50 (cat. no. 123.02D) for 5-50 ml
samples. Dynal MPC™ magnets may also
be used, except Dynal MPC™-1 (cat. no.
120.01D))
• Flow cytometry antibody reagents (optional): See www.invitrogen.com/immunology
for recommended products and protocols.
Note:
• BSA can be replaced by human serum
albumin (HSA) or 2 % FBS/FCS.
• EDTA can be replaced by 0.6 % sodium
citrate.
� Critical notes
• Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads
do not settle in the tube
• This product should not be used with
Dynal MPC™-1 (cat. no. 120.01D)
• Avoid air bubbles during pipetting
• When isolating phagocytic/adherent cells,
perform the cell isolation at low temperature (2-8°C or on ice)
• To allow bead release it is critical to use
DSB-X labeled antibodies/proteins
• For flow staining of cells after isolation use
a primary fluorescent antibody that does
not bind to the same epitope as your DSB-X
labeled antibody. Avoid using secondary
antibodies.
3. PROTOCOL
This protocol describes magnetic labeling and isolation of cells from 5x107
cells using Dynabeads FlowComp Flexi. When working with fewer cells
than 5x107, use the same volumes as indicated. When working with higher
cell numbers, scale up all reagent volumes and total volumes accordingly.
Preparations
• DSB-X label your antibodies following the procedure recommended in
the DSB-X Biotin Protein Labeling Kit (or use previously DSB-X labeled
antibodies).
• Prepare a single cell suspension of 1x108 cells/ml in Isolation Buffer.
Please visit www.invitrogen.com/cellisolation and follow our QuickLinks
for recommended sample preparation procedures.
• Prepare approximately 10 ml of Isolation Buffer per 5x107 cells.
Isolation procedure
Please see Chapter 5 (Technical Recommendations) for additional advice.
• Resuspend 5x107 cells in 500 µl Isolation Buffer and add 25 µl DSB-X
labeled antibody.
Mix well and incubate for 10 min at 2 – 8°C.
• Add 2 ml cold Isolation Buffer to wash cells, followed by centrifugation
for 8 min. at 350 x g.
• Remove and discard the supernatant.
• Add 1 ml cold Isolation Buffer to the cell pellet and resuspend.
• Add 75 µl resuspended FlowComp Dynabeads and mix well.
Incubate for 15 min at 2 – 8°C under rolling and tilting.
• Place the tube in the magnet for minimum 1 min. Carefully remove
and discard the supernatant.
• Remove the tube from the magnet. Add at least 1 ml cold Isolation
Buffer and resuspend the bead-bound cells by gentle pipetting 5 times.
• Place the tube in the magnet for minimum 1 min. Carefully remove
and discard the supernatant.
• Remove the tube from the magnet and carefully resuspend the beadbound cells in 1 ml FlowComp Release Buffer.
Incubate for 10 min. at room temperature under rolling and tilting.
• Mix the cells by gently pipetting 5 times and place the tube in the magnet
for 1 min.
• Transfer the supernatant containing the bead-free cells to a new tube
and place the tube in the magnet for 1 min to remove all residual
beads.
• Transfer the supernatant containing the bead-free cells to a new tube.
• Add 2 ml Isolation Buffer followed by centrifugation for 8 min. at 350 xg.
• Discard the supernatant and resuspend the cell pellet in preferred cell
medium. Keep the cells on 2 – 8°C until further use.
08-10-03 08.59.33
4. GENERAL INFORMATION
Manufactured by Invitrogen Dynal AS.
Invitrogen Dynal AS complies with the
Quality System Standards ISO 9001:2000
and ISO 13485:2003.
Description of Materials
FlowComp Dynabeads are uniform, superparamagnetic polymer coated beads (2.8 µm
diameter) coated with recombinant streptavidin. The FlowComp Release Buffer contains modified biotin supplied in phosphate
buffer with 0.1% BSA and 2 mM EDTA.
Storage and Stability
This product is stable until the expiry date
stated on the label when stored unopened
at 2-8°C.
Store opened vials at 2-8°C and avoid bacterial contamination.
Keep Dynabeads in liquid suspension during
storage and all handling steps, as drying will
result in reduced performance. Resuspend
well before use.
Warnings and Limitations
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.
Certificate of Analysis/Compliance is available upon request.
Material Safety Data Sheet (MSDS) is available at http://www.invitrogen.com.
Patents and Trademarks
Dynal®, Dynabeads®, FlowComp™, Dynal
MPC™ and DynaMag™ are either registered
trademarks or trademarks of Invitrogen
Dynal AS, Oslo, Norway. DSB-X™ is a trademark of Molecular Probes, Eugene, Oregon.
Any registration or trademark symbols used
herein denote the registration status of
trademarks in the United States. Trademarks
may or may not be registered in other countries.
Intellectual Property Disclaimer
Invitrogen Dynal will not be responsible for
violations or patent infringements that may
occur with the use of our products.
Warranty
The products are warranted to the original
purchaser only to conform to the quantity
and contents stated on the vial and outer
labels for the duration of the stated shelf
life. Invitrogen Dynal's obligation and the
purchaser's exclusive remedy under this
warranty is limited either to replacement, at
Invitrogen Dynal's expense, of any products
which shall be defective in manufacture, and
which shall be returned to Invitrogen Dynal,
transportation prepaid, or at Invitrogen
Dynal's option, refund of the purchase price.
110.60D.indd 2
Claims for merchandise damaged in transit
must be submitted to the carrier.
This warranty shall not apply to any products which shall have been altered outside
Invitrogen Dynal, nor shall it apply to any
products which have been subjected to misuse or mishandling. ALL OTHER WARRANTIES, EXPRESSED, IMPLIED OR STATUTORY,
ARE HEREBY SPECIFICALLY EXCLUDED, INCLUDING BUT NOT LIMITED TO WARRANTIES OF MERCHANTABILITY OR FITNESS
FOR A PARTICULAR PURPOSE. Invitrogen
Dynal's maximum liability is limited in all
events to the price of the products sold by
Invitrogen Dynal. IN NO EVENT SHALL INVITROGEN DYNAL BE LIABLE FOR ANY SPECIAL, INCIDENTAL OR CONSEQUENTIAL DAMAGES. Some states do not allow limits on
warranties, or on remedies for breach in certain transactions. In such states, the limits
set forth above may not apply.
Limited Use Label Licence
No. 5: Invitrogen Technology – The purchase
of this product conveys to the buyer the
non-transferable right to use the purchased
amount of the product and components of
the product in research conducted by the
buyer (whether the buyer is an academic or
for-profit entity). The buyer cannot sell or
otherwise transfer (a) this product (b) its
components or (c) materials made using this
product or its components to a third party or
otherwise use this product or its components or materials made using this product
or its components for Commercial Purposes.
The buyer may transfer information or
materials made through the use of this product to a scientific collaborator, provided
that such transfer is not for any Commercial
Purpose, and that such collaborator agrees
in writing (a) not to transfer such materials
to any third party, and (b) to use such transferred materials and/or information solely
for research and not for Commercial Purposes. Commercial Purposes means any
activity by a party for consideration and may
include, but is not limited to: (1) use of the
product or its components in manufacturing;
(2) use of the product or its components to
provide a service, information, or data; (3)
use of the product or its components for
therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its
components, whether or not such product or
its components are resold for use in
research. Invitrogen Corporation will not
assert a claim against the buyer of infringement of patents owned or controlled by
Invitrogen Corporation which cover this product based upon the manufacture, use or
sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in
research by the buyer in which this product
or its components was employed, provided
that neither this product nor any of its components was used in the manufacture of
such product. If the purchaser is not willing
to accept the limitations of this limited use
statement, Invitrogen is willing to accept
return of the product with a full refund. For
information on purchasing a license to this
product for purposes other than research,
contact
Licensing Department,
Invitrogen Corporation,
1600 Faraday Avenue, Carlsbad,
California 92008.
Phone (760) 603-7200.
Fax (760) 602-6500.
Email: [email protected]
5. TECHNICAL RECOMMENDATIONS
Choice of antibody
The choice of antibody clone is the most
important factor for successful cell isolation.
Please note that not all antibodies are suited
for cell isolation with magnetic beads,
although proven successful for staining.
• The time for release can be optimised in
the range of 2 – 20 minutes. 2 – 10 minutes
is usually sufficient.
• The number of washing steps (of beadbound cells) can be optimised in the range
of 1 – 5. One washing step is usually sufficient.
• The number of release steps can be optimised in the range of 1 – 3. One release
step is usually sufficient.
Please contact Invitrogen Dynal for further
technical information (see contact details).
Antibody-coating of cells
• The amount of antibody used for coating
can be optimized in the range of 0.1 – 1 µg/
106 target cells. 0.5 µg/106 target cells is
usually sufficient. Very low levels of target
cells may require larger amounts of antibody, longer incubation time or higher cell
concentration. In general, the concentration of antibody during coating of cells is a
very important factor for good results.
• Coating temperature can be varied in the
range of 0 – 37°C. 2 – 8°C is usually preferred to reduce biological activities in the
cell (e.g. enzymatic cleavage or internalization of receptors) while still keeping
coating time as short as possible.
• Coating time can be optimized in the range
of 5 – 30 minutes. 10 minutes is usually
sufficient. Coating on ice (compared to
2 – 8°C) will require longer coating time
(e.g. 20 minutes).
Storage and handling of conjugates
Please follow the recommendations in the
DSB-X Biotin Protein Labeling Kit. When
stored correctly, the conjugate should be
stable at 4°C for several months.
Cell isolation and release
• To avoid aggregation of cells it is recommended to use gamma-globulin or Fc blocking reagents.
• Cell isolation time can be optimised in the
range of 5 – 30 minutes. 10 – 20 minutes
is usually sufficient for optimal recovery.
• The recommended cell concentration
(5 x 107 cells/ml) is applicable when the
target cell content is below 50 %. If the
target cell content exceeds 50 % the cell
concentration should be reduced accordingly. Alternatively, the amount of beads
can be increased above 75 µl/ml of cell
sample. For isolation of rare cells (e.g.
stem cells from BM or leukophoresis) the
cell concentration can be increased up to
1 x 108 cells/ml.
• The temperature chosen for both cell isolation and release may affect the efficacy
of the system. The cell isolation and
release can be performed in the range of
0 - 37°C depending on needs. For isolation
of phagocytic cells (i.e. monocytes or
macrophages) keep the temperature low
since phagocytic cells may engulf beads
at temperatures above 2 – 8°C, and these
cells can not be released.
Invitrogen Dynal is a part of the Invitrogen Group.
Contact details for your local Invitrogen sales
office/technical support can be found at
http://www.invitrogen.com/contact
© Copyright 2007 Invitrogen Dynal AS, Oslo, Norway.
All rights reserved.
Revised: 10.2007
SPEC-06207
Printed: 10.2007
08-10-03 08.59.33