Version 1.0 Dec, 2019

Version 1.0 Dec, 2019

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Content

Chapter 1 Overview 2

1.1 Intended Use 2

1.2 Description 2

1.3 Workflow 3

Chapter 2 Required Materials and Equipments 4

Chapter 3 Reagent Preparation 6

Chapter 4 Blood Collection and Handling 7

4.1 Phlebotomy 7

4.2 Transportation 7

Chapter 5 Leucosep Preparation 8

5.1 Pre-Blocking of Tips or Tubes 8

5.2 Leucosep Preparation 8

Chapter 6 RBC Lysis 10

Chapter 7 WBC Depletion 11

Chapter 8 CSV Enrichment 13

Chapter 9 Cell Immunostaining 15

Chapter 10 Microscope Examination 16

Chapter 11 Maintenance 17

11.1 Histopaque® Troubleshooting 17


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Overview

1.1 Intended Use

LiquidCell™ is intended for separating circulating rare cells from

human bodily fluids utilizing functionalized magnetic beads and a

high-performance glass slide for maximal cell capture and retention.

LiquidCell™ is designed for life science and clinical research

applications supported by wide repertoire of pre-validated,

standardized GMP bioregeants targeting cancers and clinical

relevant biomarkers.

1.2 Description

LiquidCell™ is an open platform for manual processing of red

blood cell lysis, white blood cell depletion via antibody conjugated

magnetic beads, and centrifugal cytospin to capture and retain all

circulating rare cells onto a specialty-coated, glass slide for

downstream immunofluorescent and molecular staining, cell

picking, and next-generation sequencing. The glass slide is

compatible with commercial automated immuno- and in situ

hybridization stainers for automated, high-throughput

processing. Moreover, LiquidCell™ is amenable to

tyramide-based fluorescent amplification and non-fluorescent

chromogenic staining. Cross-contamination is minimized by

adopting single-use consumables.


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1.3 Workflow

Blood Collection

Leucosep Preparation

(Operating time: ~30 min)

RBC Lysis (Operating time: ~10 min)

WBC Depletion (Operating time: ~40 min)

CSV enrichment (Operating time: ~60 min)

Cell Immunostaining (Operating time: ~130 min)

Microscope Examination


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Required Materials and Equipments

Depletion Kits Catalog# Brand

CD45 Depletion Kit CSV enrichment antibody.

Kit content:

1. CD45 Magnetic AbBeads

2. RBC Lysis Buffer

3. CTC Buffer 1

4. CTC Buffer 2

5. Mouse Anti-CSV monoclonal antibody

DM0008 Abnova

3D Magnetic Separator DU0009 Abnova

Specialty-Coated Glass Slide Catalog# Brand

SuperSlide™ DL0001 Abnova

Antibody Catalog# Brand

Cell-Surface Vimentin (CSV) monoclonal antibody (FITC) DH0043 Abnova

CD45 monoclonal antibody (PE) DH0044 Abnova

CD45 monoclonal antibody PLUS (PE) DH0090 Abnova

Reagent Catalog# Brand

FcR Blocking Reagent U0318 Abnova

Staining Blocker U0330 Abnova

50X Antibody Dilution Buffer (50X ADB) U0320 Abnova

Leukocyte Aggregation Inhibitor DU0008 Abnova

Hoechst 33342 U0334 Abnova

Anti-Mouse IgG MicroBeads 130-048-402 Miltenyi Biotec

Chemical Catalog# Brand

8% Paraformaldehyde (formaldehyde) Aqueous Solution 157-8 Electron Microscopy

Science

http://www.abnova.com/products/products_detail.asp?catalog_id=DM0008

http://www.abnova.com/products/products_detail.asp?catalog_id=DU0009

http://www.abnova.com/products/products_detail.asp?catalog_id=DL0001

http://www.abnova.com/products/products_detail.asp?catalog_id=DH0043



http://www.abnova.com/products/products_detail.asp?catalog_id=U0318


http://www.abnova.com/products/products_detail.asp?catalog_id=DU0008



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UltraPure™ 0.5M EDTA, pH 8.0 15575020 Fisher Scientific

DPBS powder 21600010 Gibco

Consumable Catalog# Brand

Bovine Serum Albumin lyophilized powder A2153 Sigma-Aldrich

LS Columns 130-042-401 Miltenyi Biotec

50 mL Centrifuge Tubes 352070 Fisher Scientific

K2-EDTA Blood Collection Tube 367525 BD Biosciences

Eppendorf Micro Test Tubes 0030 121.589 Eppendorf

Histopaque®-1077 10771 Sigma-Aldrich

Leucosep® 227290 Greiner Bio-One

Cyto Funnel, 8 mL M966-8 Simport CANADA

Cover slide 0101040 Marienfeld-Superior

Equipments Catalog# Brand

Hettich Centrifuge university 320 R 1406 Hettich

Cyto rotor, 6-place 1626 Hettich

Carriers 1660 Hettich

MACS MultiStand 130-042-303 Miltenyi Biotec

Dako pen #S200230 Agilent,Dako


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Reagent Preparation

All the reagents/solutions used should be of molecular grade and

sterile-filtered!

4% PFA (4% Paraformaldehyde Aqueous Solution)

Dilute 8% paraformaldehyde aqueous solution with 1X PBS. 4%

PFA can be stored at 4°C for one week.

1X ADB

Dilute 50X ADB by adding adequate 1X PBS to 50X ADB. 1X ADB

should be kept on ice or at 4°C and used within 2 days.

MACS buffer

Prepare a solution containing PBS (pH 7.2), 0.5% bovine serum

albumin (BSA), and 2 mM EDTA

10% Glycerol/PBS

1. Dilute 1 mL of glycerol in 9 mL of PBS.

2. Sterilize the solution by passing it through a 0.22-μm filter.

3. Store in 500-μL aliquots at 4°C


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Blood Collection and Handling

4.1 Phlebotomy

Access to a peripheral vein should be performed with a 22G needle.

The first 2 mL of collected blood should be discarded to avoid

potential skin cell contamination from venepuncture. Then transfer

10 mL of blood sample in each K2-EDTA Blood Collection Tube.

Gently rotate the 10 mL blood tube end-over-end five times

immediately. Place the collected blood sample at room temperature

before use.

Do not use blood collection tubes containing fixatives to avoid

permeabilization of cell membranes.

4.2 Transportation

Blood has a maximum preservation time of 12 hours at room

temperature. For example, if the blood is drawn at 9 am in a hospital,

it should be processed before 9 pm.


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Leucosep Preparation

5.1 Pre-Blocking of Tips or Tubes

Rinse tips, pipets and tubes with CTC Buffer 1 once before use.

5.2 Leucosep Preparation

Wrap the Histopaque® -1077 stock with aluminum foil and

store at 4℃ in the dark before use. Refer to Histopaque®

Troubleshooting in Chapter 10.

Bring refrigerated 40 mL PBS to room temperature (RT)

overnight before the day of the experiment. Keep the PBS in

RT until it is used up.

5.2.1 Add 3mL Histopaque® -1077 into 15 mL Leucosep® tube

(Tube A1). Repeatly, add 3 mL Histopaque® -1077 into 15

mL Leucosep® tube (Tube A2).

5.2.2 Spin down to make Histopaque® -1077 stay in the tube

bottom, briefly

5.2.3 Dilute 7.5 mL blood sample at 1:1 ratio with 1X PBS in

Falcon® 15 mL Centrifuge Tube by using a Pipette Aid.

Invert the tube gently. The final volume is 15 mL.

5.2.4 Carefully add 7.5 mL of diluted blood sample into Tube A1

and Tube A2.

5.2.5 Centrifuge Tube A1 and A2 at 1000 xg in roomtemperture

for 10 minutes.


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After centrifugation in 5.2.5, do not proceed further if the density

gradient color and layer in the Leucosep® tube resemble the

examples below due to preexisting low density RBC or RBC

lysis in the patient’s blood sample. Low density RBC and RBC

lysis will adversely affect downstream CTC enrichment and

recovery.

5.2.6 Discard the supernatant above 5 mm PBMC layer with

suction, then transfer the PBMC layer into 50 mL tube

( Tube B, pre-blocking) with broad tip (pre-blocking)

carefully.

5.2.7 Wash Tube A1 and Tube A2 with 3 mL CTC Buffer 1, then

transfer the wash buffer to into Tube B.

5.2.8 Add CTC Buffer 1 to Tube B until 45 mL, centrifuge 300g, 5

min, discard supernatant.


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RBC Lysis

6.1 Resuspension in 1 mL CTC Buffer 1 (make sure all cell pellet

suspense in the buffer completely), add 9 mL RBC Lysis Buffer

gently, stay at RT for 3 min.

6.2 Add CTC Buffer 1 to final 45 mL, centrifuge 300g, 5 min, discard

supernatant.


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WBC Depletion

7.1 Re-suspend cells and add 2 mL CTC Buffer 1 and 4 μL

Leukocyte Aggregation Inhibitor (make sure cell pellet suspense

in buffer completely), then add 1 mL CD45 Magnetic AbBeads

(from 125 μL CD45 Magnetic AbBeads that is pre-blocked with 1

mL CTC Buffer 1 for 30 min), shaking at 125 rpm, RT, for 15 min.

7.2 Place Tube B on 3D Magnetic Separator for 2 min, transfer all

Sup. above the beads into 50 mL tube (Tube C, pre-blocking)

with 1 mL tip (pre-blocking).

***Keep tip for later usage (step 7.3 ~ 7.5.)

7.3 Re-suspend beads in 1 mL MACS Buffer by pipetting with 1 mL

tip (pre-blocking, from step 7.2) for 5 times.


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7.4 Place Tube B on 3D Magnetic Separator for 2 min, transfer all

Sup. above the beads with 1 mL tip (pre-blocking, from step 7.2)

into 50 mL tube (Tube C).

7.5 Repeat step 7.3 ~ 7.4 once.

7.6 Centrifuge 300g for 5 min, discard Sup. till approximately 0.1 mL.

7.7 Place Tube C on 3D Magnetic Separator for 2 min to remove

beads completely.

7.8 Transfer 100 μL supernatant into 15mL tube.


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CSV Enrichment

8.1 Add mouse anti-CSV mAb to 40 μg/mL and incubate at 4℃ for

20min (tap halfway).

8.2 Add 3 mL MACS buffer and centrifuge 300g for 10min. Discard

supernatant.

8.3 Resuspend cell pellet in 100 μL MACS Buffer, add 10 μL

anti-Mouse IgG MicroBeads, mix well and incubate for 20min at

4℃ (tap halfway)

8.4 Add 3 mL MACS buffer and centrifuge 300g for 10min. Discard

supernatant.

8.5 Resuspend cell pellet in 500 μL MACS Buffer

8.6 Place a LS column on MACS MultiStand, add 3ml MACS Buffer

onto the LS column.

8.7 Apply cell suspension onto LS column.

8.8 Wash LS column with 3ml MACS Buffer 3 times.

8.9 Remove LS column on 15ml tube, add 5ml MACS Buffer onto

the LS column, Immediately flush out the magnetically labeled

cells by firmly pushing the plugger into the column.

8.10 Centrifuge 300g for 10min, Discard Sup. till ~ 0.2ml.

8.11 Re-suspense the cell pellet by tapping, then add 10 mL CTC

Buffer 2.

8.12 Centrifuge 300g for 5min, discard Sup. till approximately 400 μL.

8.13 Assembly 2-well SuperSlide.

8.14 Wash 2-well SuperSlide with 1 mL PBS twice.


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8.15 Transfer 200 μL supernatant into 2-well SuperSlide with 200 μL

tip, respectively.

***Keep tip for later usage (step 8.16 ~ 8.17.)

8.16 Wash Tube C with 200 μL of CTC Buffer 2 then transfer to one of

2-well SuperSlide (from step 8.15)

8.17 Wash Tube C with another 200 μL of CTC Buffer 2 then transfer

to another one of 2-well SuperSlide (from step 8.15)

8.18 Stay at RT for 1 hr, add 60 μL of 4% PFA into each well of 2-well

SuperSlide, stay at 4 degree for overnight.

8.19 Add 73 μL of 4% PFA into each well of 2-well SuperSlide, stay at

RT for 15 min.

8.20 Cytospin of 2-well SuperSlide at centrifuge 300g, 5 min by

Hettich Centrifuge university 320 R (loosen the chamber lockers

before centrifuge).

8.21 Remove chamber.

8.22 Wash with PBS twice

8.23 Add 200 μL PBS onto each sample area, immediately.

8.24 Ready for staining.


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Cell Immunostaining

9.1 Circle sample area with DAKO pen.

9.2 Blocking with 150 μL of blocking reagent each well (65 μL 1X

ADB + 60 μL FcR Blocking Reagent + 25 μL Staining Blocker) at

RT for 20 min.

9.3 Remove blocking reagent.

9.4 Add 150 μL of antibody each well (84.3 μL 1X ADB + 25 μL FcR

Blocking Reagent + 25 μL Staining Blocker + 3 μL Cell-Surface

Vimentin (CSV) monoclonal antibody (FITC) + 11.7 μL CD45

monoclonal antibody (PE) + 1 μL CD45 monoclonal antibody

PLUS (PE) in blocking reagent) at RT for 1hr.

9.5 Wash with PBS for 3 times.

9.6 Add 150 μL 10 μg/mL Hoechst 33342 in PBS on slide, stay at RT

for 30 min.

9.7 Wash with PBS for 3 times.

9.8 Add 30 μL 10% glycerol/PBS then cover with cover slide.

9.9 Seal cover slide with nail polish.


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Microscope Examination

10.1 Fluorescent Filters & Exposures

The following microscope filters and exposures have been setup for

Nikon TiE fluorescent microscope to achieve the best staining

results. Each microscope needs be adjusted based on its

specification and staining compatibility. We highly recommend

starting with peripheral blood white blood cells and spike cells to

establish the baseline exposure and staining to avoid false positive

and false negative results.

Fluorochrome EX

(nm)

EM

(nm) Objective Filter Exposure (ms)

FITC 489 515 20X SemRock,

Cat# SpGr

400-800 ms

(no longer than 1 s)

Hoechst 33342 350 451 20X Chroma,

Cat# 49000 < 100 ms

R-phycoerythrin (PE) 565 578 20X SemRock,

Cat# SpOr

400-800 ms

(no longer than 1 s)


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Maintenance

11.1 Histopaque® Troubleshooting

Cause1 Solution

1

Blood draw n >24 hours prior to

separation

Use blood drawn from 2-6 hours prior to separation.

Blood drawn >24 hours will be more difficult to

separate, and percent recovery and viability will be

lower.

Blood has coagulated due to absence

of anticoagulant

Use of vacuum tubes with premeasured amounts of

anticoagulant is suggested. Use of a syringe

(without anticoagulant) is not recommended.

Blood has coagulated due to

incomplete mixing

Ensure vacuum tubes with anticoagulant are mixed

well after blood draw.

Blood sample is too warm and

separation attempted too soon after

blood draw

Red blood cell contamination may be a result of

blood not being at room temperature. After drawing,

the blood should be allowed to cool at room

temperature for minimum 30-45 minutes. If used

immediately after being drawn, the number of

mononuclear cells collected will be low. Separation

procedures are optimal when both blood and

Histopaque are at 18–20 °C, with an acceptable

temperature range of 18–26 °C.

Differences in donor blood physiology

(when single sample has poorer

recovery)

High lipid levels, rheumatoid factor, anemia, and

drug treatment are all possible causes for poor

separation of a specific donor’s blood. If the plasma

is not clear, this is an indication of high lipid levels.

Occlusion of white blood cells in

samples with high levels of white cells

For routine blood specimens, percent recovery will

often be higher for undiluted blood. However,

samples from donors with abnormally high white cell


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counts should be diluted to reduce the size of the

red cell aggregates and improve recovery of white

cells.

Histopaque temperature is too cold

A 100 mL or 500 mL bottle of Histopaque stored at

2–8 °C may take several hours to reach 18–20°C.

When planning to use Histopaque, we recommend

removing the Histopaque from the refrigerator the

previous day and let the bottles stand on the bench

overnight. This ensures the solution is at room

temperature and ready for use.

Incorrect centrifugation force used

The centrifuge should be checked to ensure proper

calibration. Excessive force (higher centrifugation

speed) will lower yield.

Cell pellet does not form single cell

suspension

When resuspending the cell pellet, only a small

amount of wash solution should be used. We

recommend no more than 0.5 mL wash media when

performing the procedure in a 15 mL or 50 mL

centrifuge tube. This wash media is gently rinsed

over the pellet.

Loss of cells during washing due to

adhesion to centrifuge tube wall

Use low protein binding centrifuge and

microcentrifuge tubes.

1. Histopaque® Troubleshooting Guide

https://www.sigmaaldrich.com/technical-documents/articles/biofiles/histopaque-troubleshooting-guide.html

https://www.sigmaaldrich.com/technical-documents/articles/biofiles/histopaque-troubleshooting-guide.html


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Abnova (Taiwan) Corporation Neihu Plant 9th Fl., No.112 & No.114, Jhouzih St., Neihu District, Taipei City, 114 Taiwan

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