Thermo Fisher Scientific CELLection Epithelial 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).

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 DynaMag™‑5Magnet DynaMag™‑15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

Thermo Fisher Scientific CELLection Epithelial User guide

Related papers

Other documents