isolation of human mononuclear cells from blood -...

UCAN-U 0001 Version November 01, 2011 of 8

Isolation of Human Mononuclear Cells from Blood

CMCI (Center for molecular and Cellular Intervention) University Medical Center Utrecht

Written By

Name

Mark Klein

Function

Lab manager

Date

Signature

Conformation

Name

Prof.dr. A.B.J. Prakken

Function

Co-Chair CMCI

Date

Signature

Changes from last version Date of version Paragraphs Date of version Paragraphs



Content

1. Subject .............................................................................................................................................. 3 2. Application ....................................................................................................................................... 3 3. Definitions and Abbreviations ........................................................................................................ 3 4. Principle ............................................................................................................................................ 3 5. Safety precautions ........................................................................................................................... 3 6. Reagents ........................................................................................................................................... 4

6.1 Chemicals ............................................................................................................................ 4 6.2 Basic Culture Medium .......................................................................................................... 4 6.3 Wash Medium ...................................................................................................................... 4 6.4 Freezing Medium ................................................................................................................. 5 6.5 20% AB medium .................................................................................................................. 5

7. Equipment and Accessories tools ................................................................................................. 5 7.1 Equipment ............................................................................................................................ 5 7.2 Accessories .......................................................................................................................... 5

8. Samples ............................................................................................................................................ 5 8.1 Sample Collection ................................................................................................................ 5 8.2 Sample Processing .............................................................................................................. 6

9. Procedure ......................................................................................................................................... 6 10. Processing of the Results ............................................................................................................... 7

10.1 Calculation of the counted cells ........................................................................................... 7 10.2 Errors ................................................................................................................................... 7

11. Accuracies and Precision ............................................................................................................... 8 12. Quality Control ................................................................................................................................. 8 13. Remarks ............................................................................................................................................ 8 14. Literature .......................................................................................................................................... 8



1. Subject

This Standard Operation Procedure (SOP) describes a method to isolate plasma and mononuclear cells from blood using gradient centrifugation.

2. Application

Isolation of lymphocytes and monocytes from peripheral blood using density centrifugation. 3. Definitions and Abbreviations

PBMC = peripheral blood mononuclear cells FBS = foetal bovine serum (≈ foetal calf serum) RT = room temperature P/S = Penicillin-Streptomycin Glu = L-glutamine v/v = volume/volume rpm = rotations per minute ml = millilitre DMSO = dimethylsulfoxide g/L = gram per litre

4. Principle

Defibrinated or anticoagulant treated blood or synovial fluid is layered on ficoll solution (figure 1A) and centrifuged for a short period of time. Differential migration during migration during centrifugation results in the formation of layers containing different cell types. The bottom layer contains erythrocytes which have been aggregated by the ficoll and, therefore, sediment completely through the ficoll solution. The layer immediately above the erythrocyte layer contains mostly granulocytes which at the osmotic pressure of the ficoll solution attain a density great enough to migrate through the ficoll layer (figure 1B). Because of their lower density, the lymphocytes are found at the interface between the plasma and the ficoll with other slowly sedimenting particles (platelets and monocytes). The lymphocytes are recovered from the interface and are subjected to washing steps to remove any platelets, ficoll and plasma residues (1).

Figure 1. Cell separation with Ficoll. A) Before centrifugation. B) After centrifugation

5. Safety precautions

Treat every blood and synovial fluid samples as infectious material. Wear disposable gloves.

Sample

Ficoll

Plasma

LymphocytesFicoll

Granulocytes / Erythrocytes

A B

Sample

Ficoll

Plasma

LymphocytesFicoll

Granulocytes / Erythrocytes

A B



Suma-Tab contains sodium-di-chloro-isocynate Xn, Harmful Turk solution Xn, Harmful Trypan blue T, Toxic

6. Reagents

6.1 Chemicals Reagents

Formula

Supplier

order number

Store at (°C)

Ficoll-Paque™Plus RPMI 1640 L-glutamine Penicillin-Streptomycin Foetal Bovine Serum Turk solution Trypan Blue Dimethylsulfoxide Human AB serum Coolcell

- - - - - - C34H24N6O14S4Na4 (CH3)2SO - -

Amersham Invitrogen Invitrogen Invitrogen Invitrogen Merck Invitrogen BDH Sanquin Bloodbank VWR Nederland

17-1440-02 52400-025 25030-024 15140-122 10270-106 109277 15250-061 103232J - 479-0492

RT 4 -20 -20 -20 RT RT RT -20 RT

6.2 Basic Culture Medium

RPMI 1640 supplemented with 1% v/v P/S (= 5ml) and 1% v/v glu (=5ml) Store at 4°C up to 1 month

6.3 Wash Medium RPMI 1640 supplemented with 1 v/v% P/S (= 5ml), 1 v/v% glu (=5ml) and 2% v/v FBS (=10 ml) FBS should be heat inactivated (1 hr at 56°C) and filtered through a 0.20 µm sterile filter using a 10 ml sterile syringe before usage. Store at 4°C up to 1 month.



6.4 Freezing Medium Mix 10 v/v% DMSO (5ml) with 90 v/v% FBS in a 50 ml sterile tube. Filter through a 0.20 µm filter using a 50 syringe in to a new 50 ml sterile tube. Store at 4°C up to 1 month

6.5 20% AB medium Thaw 5 ml heat inactivated human (1 hr at 56°C) and filter through a 0.20 µm sterile filter using a 10 ml sterile syringe. Add 20 ml basic culture medium and mix. Store at 4°C up to 1 week.

7. Equipment and Accessories tools

7.1 Equipment • Centrifuge Hettich Rotanta 46 (UMC# 99-000-2142) • Reichert brightline hemacytometer (Hausser Scientific Company, Horsham PA, USA) • Easypet pipet (Eppendorf, Germany, 4006173) • Pipets 10-1000 µl (Gilson, The Hague, The Netherlands)

7.2 Accessories

• 1.8 ml sterile polypropylene Nunc crytubes (Nalge Nunc, Roskilde, Denmark, 375418 • 15 ml sterile polypropylene conical tubes (Falcon/ Becton Dickinson, Erembodegem,

Belgium, 352096) • 50 ml sterile polypropylene conical tubes (Falcon/ Becton Dickinson, Erembodegem,

Belgium, 352070) • Minisart 0.20 µm single use sterile filter (Sartorius, Hanover, Germany, 16534) • 10 ml sodium-heparin vacutainer blood collection tubes (Becton Dickinson,

Erembodegem, Belgium, 366480) • Sterile 10 ml syringe (Becton Dickinson, Erembodegem, Belgium) • Sterile 50 ml syringe (Tyco Healthcare, Gosport, Northern Ireland, 1100 660131) • 2 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium,

357507) • 5 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium,



357525) • Sterile Pasteur pipettes • Disposable gloves (Kimberly Clark, Zaventem, Belgium) • Easyload 200 µl (741035) and 1000 µl (741000) sterile pipettips (Greiner Bio-one,

Germany)

8. Samples

8.1 Sample Collection Whole blood should be drawn in sodium–heparin tubes with a venoject vacuum system.



8.2 Sample Processing All handlings of the sample should be done in a biohazard safety cabinet will wearing gloves Centrifuge blood samples for 10 minutes at 150 x g (=1200 rpm) and collect 1 ml plasma in a 1.8 ml Nunc cryovial which is properly labeled. Store the plasma at –80°C. Use the remains for layering on the ficoll.

9. Procedure

1 Fill a 15 ml sterile tube with 4 ml ficoll (density = 1077 g/L) 2 Transfer blood from the collection tube in to a sterile 50 ml tube. 3 Dilute the samples with an equal volume of basic culture medium (20 ml blood with 20 ml

basic culture medium). 4 Slowly layer the diluted samples with a 10 ml sterile pipet onto the ficoll. Be careful not to

disturb or mix the ficoll sample interface. Note that a minimum of 4 ml sample is needed that can be layered onto 4 ml ficoll.

5 Centrifuge the 15 ml tubes for 20 minutes at 580 x g (= 2300 rpm), RT with a ramp-up of 9 and a break of maximum 3.

6 Fill a 50 ml with 10 ml wash medium 7 Harvest the interface (PBMC fraction) of the 15 ml ficoll tube using a sterile Pasteur pipet

into the 50 ml tube containing the wash medium. Collect as little plasma and ficoll as possible.

8 Fill the 50 ml tube containing the PBMC up to 50 ml with wash medium 9 Centrifuge 10 minutes at 280 x g (1600 rpm), RT with maximum ramp-up and brake 10 Remove the supernatant of the 50 ml 11 Resuspend the cells with a 2 ml sterile pipet 12 Remove cellular debris within the 2 ml pipet by keeping it at an angel of 90° and empty

slowly 13 Fill the tube up to 50 ml with wash medium 14 Centrifuge 10 minutes at 280 x g (1600 rpm), RT with maximum ramp-up and brake 15 Remove the supernatant 16 Resuspend the cells with a sterile 2 ml pipet

a) in 2 ml wash medium when cells are frozen b) in 2 ml 20% AB medium when cells are cultured

17 Dilute 10 µl cell suspension with 90 µl counting solution a) Use Turk solution when cells are from freshly isolated b) Use Trypan blue when viability has to be assed

18 Count cells a) Add approximately 10 µl counting-cell solution to the counting chamber b) Do not over or under fill and leave the counting chamber for one minute at RT so the

cells can settle c) Count the center 1 cubic millimeter square (each cubic millimeter equals the 25

groups of 16 squares, see figure 2). d) Count total cells per ml when using Turk solution (dark colored cells are cells with a



nucleus which is stained with Turk, erythrocytes are lysed in this solution) e) Count total live and dead cells per ml when using Trypan blue (Trypan blue is a

viable dye: living cells with an intact cellular membrane are capable of excluding the dye. Dead cells are stained dark blue. Note that erythrocytes are visible.)

f) Count a clump of cells as one. If there are many clumps resuspend the original sample additional by pipetting and recount the sample)

Figure 2. Layout of Reichert brightline hemacytometer

19 Adjust the cell concentration to 2*106 per ml with 20% AB medium when cell are being cultured and move to protocol UCAN-U 002

T-cell proliferation 20 When cells are frozen, fill the tube containing the cell suspension up to 50 ml with wash

medium 21 Centrifuge 10 minutes at 280 x g (1600 rpm), RT with maximum ramp-up and brake 22 Label sterile1.8 ml nunc cryovials 23 Remove the supernatant of the 50 ml 24 Resuspend the cells in a concentration of 10*106 per 1.5 ml in to cold (+4°C) freezing

medium (the ideal number is 10*106 per 1.5 ml. More and less cells can be frozen in the same volume but do not exceed the limit of 4 – 15 *106 cells per 1.5 ml

25 Place the Nunc 1.8 ml cryovials on ice 26 Aliquot the cell suspension at 1.5 ml per nucn cryovial 27 Place cells in –80°C freezer within 15 minutes after resuspending with freezing medium 28 Transfer the cryovials within one week from the –80°C into the liquid nitrogen tank

10. Processing of the Results

10.1 Calculation of the counted cells Number of cells per ml = Counted cells per mm2 (25 squares) * dilution (10) * 10.000 = Counted cells * 105

10.2 Errors The Isolation of cells can be disturbed by a number of factors • Too low temperature of the reagents (ficoll) or sample (<16°C) • Wrong layering of the sample (to fast) • Wrong centrifugation speed • Full break after centrifugations • Not careful collection of the interphase Furthermore clotted blood can disturb the interphase when clots are sinned through the ficoll.



11. Accuracies and Precision

Accuracy is depending on the sample, the temperature of the sample and the reagents. Ideal temperature is in between 16 and 22°C. The recovery of cells when the procedure is followed correctly: Blood ± 1.0 *106/ml depending on health status of each individual

95 ± 5% of cells present in fraction are mononuclear cells 60 ± 10% lymphocytes from original sample > 90% vitality 3 ± 2% granulocytes 5 ± 2% erythrocytes

Synovial Fluid N/A

12. Quality Control

The mononuclear fraction can be checked on purity by staining for CD15 which a granulocyte marker (see protocol UCAN-U 0003 FACS staining). The procentage of CD15 positve cells should be less the 5%.

13. Remarks

Do not close the Nunc cryovials to tight, as this will break the inner seal. Screw top firmly but stop when resistance is experienced.

14. Literature

(1) Boyum A. Separation of White Blood Cells. Nature 1964; 204:793-4.:793-794.

***END***

isolation of human mononuclear cells from blood -...

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