Cell Separation Protocol

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MANUALpluriBead® Cascade2Contents

pluriBead® Cell Separation Technology

3pluriBead® in Detail4
Cell Separation Protocol
Required Materials

5

1. Sample Preparation and Target Binding6

2. Washing8

3. Detachment10
Troubleshooting11

Warnings & Limitations

This product is developed for scientific use only. Itmust not be used for diagnostic or therapeutic purposes in animals or men.
This product is developed for laboratory use only.
Users must follow the appropriate laboratory guidelines.
pluriBead® suspension contains conservatives such as
sodium azide which are toxic if ingested. Thus, pipetting by mouth must be avoided.

General Advice

Make sure that the kit box is intact and that buffers
have not leaked. Do not use a kit that has been damaged.
When working with multiple samples, avoid transferring samples to the wrong mixing containers, strainers or tubes. Ensure that all containers, strainers ortubes are properly labeled using a permanent pen.
Unless otherwise indicated, all steps of this protocol
should be carried out at room temperature.

Safety Advice

When handling with biological and chemical materials, always wear a laboratory coat, disposable gloves
and protective goggles to prevent infections (e.g. byHIV or hepatitis B viruses) and to avoid injuries. In
case of contact between gloves and sample, change
gloves immediately.

Storage & Stability

pluriBead® suspension, Wash Buffer, Buffer A (Stabilization Buffer), Buffer B (Incubation Buffer) and Buffer
C (Detachment Activation Buffer) should be stored at
2-8°C. Buffer D (Detachment Concentrate) must be
freezed at -20°C.
Properly stored and handled, pluriBead® suspension
and buffers are stable until the expiration date statedon the label. Deviant storage conditions will lead topoorer separation results.
Thoroughly resuspend pluriBead® suspension before
use. Avoid bacterial or fungal contamination of thepluriBead® suspension and provided buffers.

Preventing Cell Stress & Contamination

Whenpipetting
thesample
intothemixing container, avoid the contamination of the containers rim.
Always change pipette tips between liquid transfers.
Use filter pipette tips.
Avoid touching the mesh of the strainer with the pipette tip.
Avoid any unnecessary contact with the inner sterile
surfaces of the kit components. In particular, the surfaces of the mixing container and the strainer should
remain sterile to avoid cross contamination.

pluriBead® Cell Separation Technology

3

pluriBead® Technology

pluriBead® uses non-magnetic monodispersed microparticles (beads) for the separation of cell mixtures. Their surface is coated with monoclonal antibodies (mAb) directed against specific structureson the target cell surface.
During incubation, the target cells in suspension will bind to the pluriBeads® and can be separated
afterwards by a pluriStrainer® (size exclusion) from the suspension. The beads are larger than thecells and thus cannot be phagocytized by them.
Pretreatment of the blood (e.g. densitiy centrifugation, erythrolysis or other target concentration)
is not required.
pluriBead® should be used for research use only.

pluriBead® Principle
Raw Material
A – pluriBeads® with target-specific mAb coating.
B – Sample material (whole blood, buffy coat orother cell suspension) containing desired targetcells.

AB

1. Incubation
Mix and gently incubate sample and pluriBeads®
at room temperature. The target cells bind to thepluriBeads®.
2. Washing
The target cells – now bound to the beads –will be isolated from the sample material by sieving. Bead-bound targets remain on the strainer
while all other cells run into a tube beneath.
3. Detachment
Target cells are detached from the beads with adetachment buffer directly on the strainer. They
are then washed into a fresh tube while thedepleted beads remain on the strainer.

Typical pluriBead® Cell Separation Profile: Example CD8

FSC/SSC analysis ofisolated population
Histogram of whole blood before (top)and after (below) depletion

Fluorescent labeledisolated population

Apoptosis staining ofisolated population

pluriBead® in Detail4

pluriBead® Particles - Phase Contrast

M-pluriBead®

S-pluriBead®

Cells after detachment

Living cells stained with Calcein AM (green)
Dead cells stained with Propidium Iodide (red)pluriBead® Size
Specification

S-pluriBead®

M-pluriBead®
AA

A

A

A
AAAAAAA

Illustration

AAAAAAAAAAAA

A - target cellA - target cell

pluriBead® Size

32 µm

65 µm

Maximum isolated cells
per separation

1x 1075x 107

Maximum bead suspension perpluriStrainer

400 µl1,000 µl

Recommended application

medium number (≤2x10 ) of targets,rare cells, andcirculating tumor cells (CTC)

Sample material

whole blood, tissue, PBMC, cell culture,
buffy coat, liquor

pluriBead® material

Polystyrene

Polystyrene

200 µl500 µl

6Minimum sample volume

large number of targets in a sample(e. g. buffy coat)
buffy coat, whole blood*, tissue, PBMC,
cell culture* only recommended for cells with highconcentration (e.g. granulocytes)

If you have problems choosing the right pluribead® size use the interactive selection
guide
http://pluriselect.com/selection-guide.html

Required Materials

5pluriBead® Suspension
pluriBeads

PictureS-pluriBead® suspension purple cap
M-pluriBead® suspension blue cap

Description
S

Storage Conditions

Catcher particles labeled withspecific antibodies

MStore at 4-8°C

S-pluriBead® Reagent Kit & M-pluriBead® Reagent Kit
Kit Components

PictureDescription

Storage Conditions

Wash buffer

Buffer for washing steps. Dilute
before use.

Store at 4–8°C

Buffer A

Chelating agent for preserving
blood sample

Store at 4–8°C

Buffer to increase density ofsample esp. for PBMC isolation

Store at 4–8°C

Add 200 µl to detachment concentrate vial for its activation

Store at 4–8°C

Detachment of target frompluriBeads

Store at –20°C(10x Stock Solution)(Stabilization Buffer)
Washbuffer

Buffer B

Buffer A

Buffer B

Buffer C(Incubation Buffer)

Buffer C(Detachment Activation Buffer)

Buffer D-20°C

Buffer D(Detachment Concentrate)!

Strainer for separating
pluriBeads with target cells fromsample and for sample pre-filtration

Room Temperature

Connector Ring incl. LuerLock

Connection to 50 ml tube. Essential for detaching the pluriBeads®

Room Temperature

FunnelSupports sample load onto
pluriStrainer®

Room Temperature

S-pluriStrainer® S / 30 µm(purple, max. load: 400 µl
S-pluriBead® suspension)

M-pluriStrainer® S / 60 µm(blue, max. load: 1000 µl
M-pluriBead® suspension)Additional required materials for working with pluriBead®
Mixer*

Gloves

Pipettes

Tubes

Centrifuge

15
1450

131211 40103098720

6541010‘
210.5

1.0

2.0

No orbital shakers or*

1.53300 x g

6

1. Sample Preparation and Target Binding

1.1 Preparation of Buffers (before the start of the separation)!

• Bring all provided reagents to room temperature.
(For the detachment concentrate: Do not use a 37°C water bath or incubator)• Dilute 10x Wash Buffer (sufficient for 1,.000 ml Wash Buffer)with sterile high-purity water (E.g. dilute 100 ml 10x Wash Buffer with 900 ml sterilwater)

Room temperature• Add 200 µl Buffer C into 1 vial of Buffer D (1.8 ml).

1.2 Preparation of Sample Material
Whole BloodTissue/PBMC• Add 50 µl of• Prepare a single cellprovided Buffer A suspension.
per 1 ml sample. Take up the cell pellet in500 µl Buffer B and 500 µl• CD14: Before separ- Wash Buffer.
ating CD14+ cells Adjust the concentration offrom a sample, re- cells at max. 5x106 targets
move sCD14! (see p. per 1 ml buffer.
11)• Pre-filter sample• Mouse whole blood: with S-pluriStrainer®
Pre-filter sample to to remove aggregates.
remove aggregates.

Buffy Coat
• Add 50 µl of
provided Buffer A per
1 ml sample.
• Pre-filter buffy coat.
Attach a provided strainer to asterile 50 ml centrifuge tube.
Place a provided funnel on top.
Carefully pour your sample intothe funnel.
If the buffy coat clogs thestrainer, prepare first PBMC or
use another buffy coat!

• Alternatively use
Buffy Coat Add-On(Order.No.: 01-00600-10)

1.3 Binding of Targets• Thoroughly resuspend pluriBead® suspension by vortexing the tube.
• Add S-pluriBead® suspension, M-pluriBead® and sample into a sterile mixing
tube:
Whole Blood:

40 µl S-pluriBead® suspension per 1 ml
100 µl M-pluriBead® suspension per 1 ml

Other samples:

40 µl S-pluriBead® suspension per 1x106 targets
100 µl M-pluriBead® suspension per 1x106 targets

50

40

30

2,020

1,51,00,510

Recommended tube sizes:
Sample + pluriBeads® 0.2–2 ml: 2 ml tube (round bottom)
Sample + pluriBeads® 2–6 ml: 15 ml tube or use the blood collection tube
Sample + pluriBeads® 6–50 ml: 50 ml tube

Incubation

7• Incubate sample up to 30 min. pluriBead® must remain in suspension.
3D platform rotator

3D platform rotator

RotatorRoll Mixer

3D platform rotator

Roll Mixer
pluriPlix®
(by pluriSelect)

RotatorHorizontal roller mixer Rotator mixer(with tilting function)

10–15 rpm/7.5° angle 10–15 rpm
Tube Mixer

Tube Mixer

Laboratory rocker

Orbital shakerRotatorRoll Mixer

Tube Mixer

For optimal results, it is important that the sample material is mixed gently! Since the particles are larger than the cells, the particles must remain
in suspension during the incubation period and may not settle, so that thecells can bind to the particles.
While incubating the sample, unfreeze the detachment buffer according
to the amount of your sample material and pre-warm the washing buffer
to room temperature. Do not use a 37°C water bath or an incubator.

82. Washing• Attach a S-pluriStrainer® to a sterile 50 ml centrifuge tube.
The bigger opening of the strainer must be on the top.
• Attach an M-pluriStrainer® onto S-pluriStrainer®.
• Optional for sample more than 4ml: Place the supplied funnel on topof the strainers and maximum application volume of liquid on the strainer
increases to 15 ml.
• Add 1 ml Wash Buffer (diluted, see step 1.1) to equilibrate the strainerand support separation.
Use one tube and strainer stack for each:• max 160 µl S-pluriBead® - or - max 400 µl M-pluriBead® used insample• Pour sample-bead-suspension onto the M-pluriStrainer®.
Unbound cells run through the strainers into the centrifuge tube, the bead-bound target cells remain on the strainers.
The flow-through can be used for further cell isolation.

washingbuffer• Recommendation: Use a 5 ml pipette (no serological pipette).
• Wash off the bead-sample traces from the funnel in 2 ml steps*
and discard the funnel.
• Wash the top strainer sufficiently in 2 ml steps with Wash Buffer.
• Wash in a circular motion, avoid washing the middle of the strainer
only.
• Take off the M-pluriStrainer® and attach it to a sterile 50 ml centrifugetube. Then, wash both strainers with 5 x 2 ml wash buffer each.
Do work fast enough to prevent the cells on both sieves from drying out.
Watch out the tubes beneath the strainers contains no morethan 45 ml of sample/Wash Buffer! Use several tubes if necessary.
The bead-bound target cells on the strainer are now ready for further use.

*Note: Wash until you do not see any red spots any more, plus 3 additional washing steps.

2. Washing

9After washing, erythrocytes or other unwanted material
might still adhere to the inner surface of the strainer. To avoid target
contamination with these during later steps of the protocol, you can also
wash the inner and outer surface as well as the bottom of the strainer.
When adding Wash Buffer with too much pressure, targets on the strainer
might be splashed away.

NoticeTarget lysis for protein or DNA/RNA analysis
For rapid and consistent results in protein or gene expression analysis,
lyse the targets while they are still attached to the beads.
• Attach the connector tightly to a new 50 ml centrifuge tube.
• Close the Luer-Lock and attach the strainer with the isolated target cells.
Caution: Do not tilt the strainer!
• Add lysis buffer according to the manufacturers direction.

3. Detachment

10

3. Detachment(Target cells will be released from the pluriBeads into the tube.)• Attach two Connector Rings straight and tight to two new 50 ml tubes.
• Attach and close the Luer-Locks.
• Attach both strainers with the isolated target cells to the Connector
Rings. Caution: Fit even and do not tilt the strainers!
• Add 1 ml Wash Buffer along the wall of the strainer.
Optional check 1 – Check if the target cells are bound to the beads.
• Resuspend the beads by gently moving the strainer in a circular way.
• Retain a small amount of sample (10 μl) and place it on a microscopeslide or in a microwell plate.
• Check if the target cells are bound to the beads.
• Add 1 ml activated Buffer D (200 µl Buffer C into 1 vial Buffer D) along
the wall of each strainer.
• Swirl the sample gently and incubate it for 10 min at room temperature.
Gently move each strainer in a circular way every 2 min. Do not pipette!
If liquid drops through the strainer, check whether the Luer-Lock is closed
and the strainer is placed correctly. It is no problem though if 100–200 µl
of liquid run through the strainer during the 10 min detachment time.
• After incubation, add 1 ml Wash Buffer on each strainer.
• Separate the cells from the beads by pipetting up and down the sample
on each strainer with a 1 ml pipette (10 times).
Avoid air bubbles and do not touch the mesh with the pipette.

10x ↑↓

Optional check 2 – Check if the cells are released from the beads.
• Retain a small amount of sample (10 μl) and place it on a microscopeslide or in a microwell plate.
• Check under the microscope if the cells are released from the beads.
• If the beads are still rosetted with cells, extend incubation time for another5 min and repeat pipetting up/down the sample on the strainer (10x).
• Open Luer-Lock. The detached cells now run into the 50 ml centrifuge
tube, pluriBeads® remain on each pluriStrainer®.
• Wash each strainer 10 times with 1 ml Wash Buffer or cell culture
medium. Wait until buffer has drained off, before adding new buffer.
• Remove connectors and strainers from the tube by lifting the connector
with the thumb. Discard both connector and strainer.
50

4030

20

1015
1410‘

13121110
98
7
65
43
2
1300 x g• For centrifugation, pour the suspension with the detached cells into afresh 15 ml tube. (Larger tubes will lose approx. 30% of your targets!)• Centrifuge the cells 10 min at 300 x g without brake.
• Carefully remove the supernatant with a pipette down to 500 µl.
(Pouring off the supernatant will lose approx. 20% of your targets!)• Transfer the cells into Wash Buffer or medium of your choice. The cells arenow separated from the beads and can be used for further experiments.

Troubleshooting

11Common Error Sources
Target Yield

40 μl S-pluriBead® suspension can bind up to 1x106 target cells. Thisdepends on the existing amount of target cells in the sample material, thedensity of receptors on target cells and an optimal mixing of thepluriBeads® with the sample.
The initial concentration of leukocytes in whole blood can be determinedby using a hemocytometer or by hand with a counting chamber and Turk‘ssolution.

Monocytes fromWhole Blood

When separating CD14 positive cells from a sample, remove sCD14 first!
1. Dilute whole blood with Wash Buffer (1 ml whole blood + 2 ml Wash Buffer).
2. Centrifuge 10 min at 300 x g (no brake).
3. Detach plasma and supernatant to approx. 1 cm above the blood.
Do not pipette too closely to the buffy coat layer to avoid pipetting cells!
4. Repeat step 1 to 3.
Use the concentrated cells for the separation of CD14+ cells but calculate
the pluriBead® volume according to the starting sample volume.

Tissue/PBMC

Detach adherent cells from the tube surface, or rather isolate tissue cells,
very gentle. Thereto, keep the digestion time as short as possible. Long
reaction times damage the cell surface receptors and thus reduce theefficiency of pluriBead®.
Stop digestion by adding complete medium and separate the cells fromone another by pipetting. Afterwards, transfer the cells into a centrifugetube and pelletize the cells.
To avoid cell aggregations, you can attach a strainer to the centrifuge tube.
While the single cells pass through, cell aggregations are held back by thestrainer.

Detachment

When adding the activated Buffer D to your bound targets, take care to notpour buffer into the middle of the strainer. Rather pour it onto the inner rimof the strainer so that the sample is swirled.
When separating the cells from the beads, do pipette carefully but not too
cautiously. Rude pipetting will result in air exchange and dripping of thebuffers. Too cautious pipetting, however, will not separate all targets fromthe catcher particles. As a result, your yield will be low.

countsPurity Analysis

For cell analysis always use CD45 for the staining of Leucocytes isolated
from whole blood, buffy coat or PBMC. That will help to exclude unwanted
erythrocytes (RBC), platelets and debris for the analysis.
* Platelets, red blood cells and debris.

*CD45

All buffers and consumables can also be ordered individually fromwww.pluriselect.com.
BuffersProductOrder No.

Size

Buffer A

60-00070-12

10 ml

Buffer B

60-00060-12

10 ml

Buffer C

60-00045-12

10 ml

Buffer D

60-00040-12

10 x 1.8 ml

Wash buffer

60-00080-10

100 mlOrder No.

Size

S-pluriStrainer®

43-50030-03

25 pcs.

M-pluriStrainer®

43-50060-03

25 pcs.

Connector Ring

41-50000-03

25 pcsFunnel42-50000-03

25 pcs

Buffy Coat Add-On

01-00600-10

1 Kit(Stablization Buffer)(Incubation Buffer)(Detachment Activation Buffer)(Detachment Concentrate)(10x Stock Solution)

Consumables
Product© pluriSelect Life Science 2016
Additional Support:
http://pluriselect.com/video-tutorials.html
support@pluriselect.com
Manual_pluriBead_Cascade_EN_V5-7