Thermo Fisher Scientific CELLection Epithelial User guide

Type
User guide
CELLection Epithelial Enrich Kit
Catalog Number16203
Pub.No. MAN0026583 Rev. A.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
The CELLection Epithelial Enrich Kit is intended for isolation of human epithelial cells directly from whole blood, bone marrow,
mononuclear cell (MNC), or tissue digests.
CELLection Epithelial Enrich Dynabeads beads are uniform, superparamagnetic polymer beads (4.5 μm diameter) coated with a
monoclonal mouse IgG1 antibody (Ber-EP4) via a DNA linker to provide a cleavage site for cell release. Ber-EP4 is an anti-EpCAM
(epithelial cell adhesion molecule) antibody with the same reactivity as HEA125, and is specific for two lycopolypeptide membrane
antigens expressed on most normal and neoplastic human epithelial cells. The Releasing Buer Component I contains freeze-dried Turbo
DNase and needs to be reconstituted in Releasing Buer Component II prior to use.
The enriched cells are bead-free, and may be cultured directly and used for immunocytochemical staining. For depletion of human
epithelial cells or positive isolation for downstream molecular analysis, we recommend using Dynabeads Epithelial Enrich beads.
CELLection Epithelial Enrich Dynabeads beads are mixed with the cell sample in a tube. The Dynabeads magnetic beads bind to the
target cells during a short incubation, and the bead-bound cells are separated by a magnet. The beads are removed from the cells using
the Releasing Buer Component 1 (Turbo DNase) (see Figure1).
1
2
3
4
5
Figure1Epithelial tumor cell enrichment
1Starting sample: whole blood, bone marrow, MNC, or tissue digests.
2Add CELLection Epithelial Enrich Dynabeads beads.
3Isolate epithelial cells using a magnet. Remove supernatant.
4Add Releasing Buer to detach beads from cells.
5Viable epithelial tumor cells for culture or staining.
USER GUIDE
For Research Use Only. Not for use in diagnostic procedures.
Contents and storage
The kit contains reagents sucient for 100 mL of whole
blood/buy coat or approximately 2 x 109 of mononuclear cells.
Table1CELLection Epithelial Enrich Kit (Cat.No.16203)
Contents Amount Storage
CELLection Epithelial Enrich
Dynabeads beads 5mL
2°C to 8°CReleasing Buer Component 1
(Turbo DNase) 2vials
Releasing Buer Component 2 2mL
Required materials not supplied
Magnet (DynaMag portfolio). For recommendations, see
www.thermofisher.com/magnets.
Mixer or shaker (e.g., HulaMixer Sample Mixer).
Buer 1: Ca2+ and Mg2+ free PBS supplemented with 0.1%
BSA, pH7.4
Buer 2: Ca2+ and Mg2+ free PBS with 0.1% BSA and 0.6%
Na-citrate or 2 mM EDTA.
Note: BSA can be replaced by human serum albumin (HSA)
or fetal calf serum (FCS).
Buer 3: RPMI 1640 with 1% fetal calf serum (FCS), 1 mM
CaCl2 and 5 mM MgCl2, pH 7.0–7.4.
Procedural guidelines
IMPORTANT! Visit http://www.thermofisher.com/
samplepreparation for recommended sample preparation
procedures.
Use a mixer that provides tilting and rotation of the tubes to
ensure that Dynabeads beads do not settle at the bottom of
the tube.
Avoid air bubbles (foaming) during pipetting.
Carefully follow the recommended pipetting volumes and
incubation times.
Keep all buers on ice throughout the procedure.
For optimal DNase activity, ensure that the pH in Buer 3 is
7.0–7.4.
Samples from healthy donors spiked with epithelial cells
(e.g. SW480, ATCC no. CCL-228) can be used as a positive
control for the enrichment protocol.
Wash Dynabeads beads
1. To resuspend the Dynabeads beads in the vial, vortex for >
30 seconds or tilt and rotate for 5 minutes.
2. Combine equal volumes of Dynabeads beads and Buer 1
in a tube, then vortex to resuspend the beads. See “Volumes
for isolation of human epithelial cells from MNC or whole
blood/buy coat” on page4.
3. Place the tube in a magnet for 1 minute, then discard the
supernatant.
4. Remove the tube from the magnet, then resuspend the
washed Dynabeads beads in the same volume of Buer
1 as the initial volume of Dynabeads beads. See “Volumes
for isolation of human epithelial cells from MNC or whole
blood/buy coat” on page4.
Release buer preparation
1. For each vial of freeze-dried Turbo DNase, transfer 300 μL
from the Releasing Buer Component 2 to each vial of
Releasing Buer Component 1 (Turbo DNase).
2. Dissolve the enzyme gently by tilting gently or pipetting up
and down.
IMPORTANT! Vigorous mixing will destroy the enzyme.
3. Aliquot the reconstituted Releasing Buer into suitable
portions.
Store at –20°C. Thaw immediately before use and keep on ice
once thawed. Thawed Releasing Buer can be re-frozen once.
Prepare sample
Wash whole blood and bone marrow
The following procedure is important for removal of interfering
factors.
1. Dilute the whole blood or bone marrow in Buer 2 (1:1).
2. Centrifuge at 600 × g for 10 min at room temperature.
3. Discard the plasma fraction/upper layer.
4. Resuspend to the original volume in Buer 2 at 2°C to 8°C.
Treat bone marrow with DNase
The following procedure is important for removal of interfering
DNA.
2CELLection Epithelial Enrich Kit User Guide
1. Dilute washed bone marrow in Buer 3 (1+4).
2. Add 120 IU/mL of DNase (not supplied).
3. Incubate for 30 minutes at room temperature with gentle
tilting and rotation.
4. Centrifuge at 600 × g for 10 minutes at room temperature,
then discard the supernatant.
5. Resuspend cells in the same volume of Buer 2.
6. Repeat step4.
7. Resuspend at 2 × 107 cells/mL in Buer 2 at 2°C to 8°C.
Prepare MNC from whole blood, buy coat, or bone
marrow
Follow procedural guidelines when preparing MNC.
Resuspend MNC at 2 × 107 cells/mL in Buer 2 at 2°C to 8°C.
Enrich tumor cells from MNC or whole
blood/buy coat and release tumor cells
Start with 1 mL (2 × 107) MNC or 5 mL whole blood or buy coat.
This is scalable from 2 × 107– 5 x 108 MNC or 5–50 mL whole
blood or buy coat.
When working with smaller volumes than shown for small-scale
(1X) isolation, use the same volumes as indicated for small-scale
(1X) isolation. When working with larger volumes, scale up all
reagent and volumes accordingly. See “Volumes for isolation of
human epithelial cells from MNC or whole blood/buy coat” on
page4.
1. Add 50 µL Dynabeads beads to 1 mL MNC, or 250 µL
Dynabeads beads to 5 mL whole blood or buy coat.
2. Incubate for 30 minutes at 2°C to 8°C with gentle tilting and
rotation.
3. Place the tube in a magnet for 2 minutes.
4. While the tube is still in the magnet, carefully remove and
discard the supernatant.
5. Remove the tube from the magnet and add 1 mL Buer (for
MNC) or 5 mL Buer (for blood or buy coat).
6. Pipet 2–3 times or vortex 2–3 seconds, then place the tube
in a magnet for 2 minutes.
7. To wash the bead-bound cells, repeat step4–step6 twice.
Note: This step is critical to obtain a high purity of isolated
cells.
8. Resuspend the bead-bound cells in 200 µL Buer 3
preheated to 37°C.
Note: Before transferring the released cells to a new tube,
pre-coat the tubes for 5 minutes using Buer 3.
9. Add 4 µL reconstituted Release Buer (DNase).
10. Incubate for 15 minutes at room temperature with gentle
tilting and rotation.
11. Pipet thoroughly with a 100–200 µL pipette at least 5–10
times to maximize cell release.
Note: Avoid foaming.
12. Place in a magnet for 2 minutes, then transfer the
supernatant with released cells into a pre-coated tube.
13. Resuspend the bead fraction in 200 µL Buer 3, then repeat
step11 and step12.
Note: The cells are now bead-free and ready for use in any
downstream applications.
CELLection Epithelial Enrich Kit User Guide 3
Volumes for isolation of human epithelial cells from MNC or whole blood/buy coat
Step Step description Small scale (1X) Large scale (10X)
Recommended tube size 5mL 15mL
Recommended magnet DynaMag5Magnet DynaMag15Magnet or
DynaMag-50Magnet
step1 Volume of MNC
Volume of blood or buy
1mL
5mL
10mL
50mL
step1 Dynabeads beads 50µL for MNC
250µL for blood/buy
500µL for MNC
2.5mL for blood/buy
step5–step7 Wash beads (Buer 1) 1mL × 3 for MNC
5mL × 3 for blood/buy
10mL × 3 for MNC
30mL × 3 for blood/buy
step8 Resuspend beads (Buer 2) 200µL 2mL
step9 Release cells (Release Buer) 4µL 40µL
step13 Collect residual cells (Buer 3) 200µL × 2 2mL × 2
Related products
Unless otherwise indicated, all materials are available through
thermofisher.com.
Product Cat. No.
DynaMag5Magnet 12303D
DynaMag15Magnet 12301D
DynaMag-50Magnet 12302D
Dynabeads Epithelial Enrich beads 16102
HulaMixer Sample Mixer 15920D
Limited product warranty
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products as set forth in the Life Technologies' General Terms
and Conditions of Sale at www.thermofisher.com/us/en/home/
global/terms-and-conditions.html. If you have any questions,
please contact Life Technologies at www.thermofisher.com/
support.
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The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No.MAN0026583
Revision Date Description
A.0 21 April 2022
The kit contents, procedural guidelines, and manufacturing site were updated in the user manual.
The user manual was re-structured and edited to meet current style guidelines.
003 15 June 2012 New manual for CELLection Epithelial Enrich Kit.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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21 April 2022
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Thermo Fisher Scientific CELLection Epithelial User guide

Type
User guide

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