Ficoll-Paque PLUS protocol.pdf

Ficoll-Paque PLUSInstructions 71-7167-00 AG Cell preparationGE HealthcareFicoll-PaqueIntended UseFor in vitro isolation of lymphocytes.Introduction and Summary of the MethodThere is a need for a rapid,simple and reliable method of isolating lymphocytes from whole blood in such areas of scientific endeavor as histocompatibility testing,in vitro cellmediated immunity assays,and other procedures requiring pure lymphocyte preparations.Early techniques for separating lymphocytes from other blood cell types involved mixing the blood with some erythrocyte aggregating agent,thereby causing the erythrocytes to clump and sediment to the bottom of the tube.The lymphocytes could then be collected from the upper part of the tube(1,2).The disadvantages of these techniques are that lengthy,repeated procedures are required to obtain purified lymphocyte suspensions,and the yield of recovered lymphocytes is low.Byum noted that the low viscosity of FicollTM,compared to the other polymeric erythrocyte aggregating agents,makes it possible to devise a lymphocyte isolation procedure involving a short,low speed centrifugation(3).A solution of Ficoll and sodium metrizoate of the proper density and osmotic strength was placed in a centrifuge tube.Blood was layered on top,and the two-phase system was centrifuged at a low speed for a short time.The erythrocytes and granulocytes sedimented to the bottom of the tube,and the purified lymphocytes could be collected from the interface between the two phases.Other authors,using slight modifications of this procedure,have p.2 Contents 1.Principle of the Procedure 3 2.Reagents 4 3.Precautions 4 4.Storage 4 5.Indications of Instability 4 6.Specimen Collection and Handling 4 7.Procedure 5 8.Parameters of the Method 5 9.Preparation of Reagents 610.Test Protocol 711.Expected Value 1012.Factors Affecting Lymphocyte Isolation by the Ficoll-Paque PLUS Procedure 1013.How Supplied 1114.Ordering Information 1115.References 12p.3emphasized that the Ficoll-sodium metrizoate centrifugation procedure is an easy one-step,rapid,reproducible method for the preparation of viable lymphocytes in high yield(47).Sodium diatrizoate has been successfully substituted for sodium metrizoate in this procedure by numerous workers(811).Ficoll-PaqueTM PLUS provides a sterile,ready-to-use Ficoll sodium diatrizoate solution of the proper density,viscosity,and osmotic pressure for use in a simple and rapid lymphocyte isolation procedure.Ficoll-Paque PLUS also provides the additional assurance of low levels of endotoxin.1.Principle of the ProcedureThe following principles apply for Ficoll-Paque PLUS.Defibrinated or anticoagulant-treated blood is layered on the Ficoll-Paque PLUS solution and centrifuged for a short period of time.Differential 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-Paque PLUS.Th layer immediately above the erythrocyte layer contains mostly granulocytes which at the osmotic pressure of the Ficoll-Paque PLUS solution attain a density great enough to migrate through the Ficoll-Paque PLUS layer.Because of their lower density,the lymphocytes are found at the interface between the plasma and the Ficoll-Paque PLUS with other slowly sedimenting particles(platelets and monocytes).The lymphocytes are then recovered from the interface and subjected to short washing steps with a balanced salt solution to remove any platelets,Ficoll-Paque PLUS and plasma.Since this method can be adapted to very small volumes of blood,it is particularly suitable for isolation of lymphocytes where only limited quantities of blood are available,such as from cadavers and small children.p.4 2.ReagentsEach 100 ml contains Ficoll PM400,5.7 g,Diatrizoate Sodium,9.0 g,with Edetate Calcium Disodium in Purified Water U.S.P.3.PrecautionsThis product is intended for in vitro isolation of lymphocytes for research applications.4.StorageFicoll-Paque PLUS should be stored between 4 C and 30 C and protected from direct light.5.Indications of InstabilityDeterioration of the Ficoll-Paque PLUS is indicated by the appearance of a distinct yellow color or particulate material in the clear solution.6.Specimen Collection and HandlingFresh blood should be used to ensure high viability of isolated lymphocytes.Prepare sample at 1820 C as follows:1.To a 10 ml test-tube add 2 ml of defibrinated or anticoagulant-treated blood*and an equal volume of balanced salt solution.(Final volume 4 ml.)2.Mix by drawing the blood and buffer in and out of a Pasteur pipette.*Anticoagulants:Heparin,EDTA,citrate,acid citrate dextrose(ACD)and citrate phos-phate dextrose(CPD)may be used.Defi brinated blood requires no anticoagulant.p.57.ProcedureMaterials ProvidedSee under”Reagents”.Materials Required but not ProvidedItem No.SizeTest tubes*2/sample 10 mlBalanced salt solution 20 ml/sample Pasteur pipettes*1/sample 3 mlCentrifuge tubes*2/sample 15 ml;internal diameter 1.3 cmCentrifuge with swing-out rotor capable of producing 60400 g and maintaining 1820 C temperatureSyringe with needle Silicone solution,1%Distilled water *Tissue culture plasticware or pretreated glassware.Preparation of glassware:All glassware which comes in contact with the sample should be siliconized before use.The glassware should be immersed in a 1%silicone solution for 10 seconds,washed with distilled water(6 times)and then dried in an oven.8.Parameters of the MethodSample volume:4 ml totalDefibrinated or anticoagulant-treated blood 2 ml Balanced salt solution 2 mlLarger blood samples:Larger volumes of blood may also be processed with the same efficiency of separation.This is effected by increasing the diameter of the centrifuge tube while maintaining approximately the same Mix p.6 height of the Ficoll-Paque PLUS(2.4 cm)and of blood sample(3.0 cm)in the centrifuge test tube(1).Smaller blood samples:Smaller quantities of blood can be processed rapidly by a modification of the recommended procedure(2).9.Preparation of ReagentsBalanced Salt Solution.At least 20 ml for each sample to be processed.The balanced salt solution may be prepared from two stock solutions,A and B.Solution A Conc.g/lAnhydrous D-glucose 0.1 percent 1.0CaCl2 2H2O 5.0 10-5 M 0.0074MgCl2 6H2O 9.8 10-4 M 0.1992KCl 5.4 10-3 M 0.4026TRIS 0.145 M 17.565Dissolve in approximately 950 ml distilled water and add 10 N HCl until pH is 7.6 before adjusting the volume to 1 litre.Solution B Conc.g/lNaCl 0.14 M 8.19To prepare the balanced salt solution mix 1 volume Solution A with 9 volumes solution B.Prepare the solution fresh each week.Other standard salt solutions may be used.p.710.Test ProtocolProcedure for isolation of lymphocytes.1.Invert the Ficoll-Paque PLUS bottle several times to ensure thorough mixing.A.For withdrawal of Ficoll-Paque PLUS by syringe.Fig 1.Snap-off the polypropylene cap(Fig 1).Insert the syringe needle through the septum.Inject air from the syringe to equalize pressure.Invert the bottle(Fig 2)and withdraw the required volume of Ficoll-Paque PLUS.B.For withdrawal of Ficoll-Paque PLUS by pipette.100 ml bottles Remove the snap-off polypropylene cap.Lift the alu-minium ring.Pull off the metal seal.Remove the silver ring.Remove the rubber septum.Using aseptic techniques,withdraw the required volume of Ficoll-Paque PLUS.500 ml bottles Remove the snap-off polypropylene cap.Lift the alumini-um ring.Pull off the metal seal.Remove the silver ring.Remove the rubber septum.p.8 Fig 2.Using aseptic techniques,withdraw the required volume of Ficoll-Paque PLUS.2.Add Ficoll-Paque PLUS(3 ml)to the centrifuge tube.3.Carefully layer the diluted blood sample(4 ml)on Ficoll-Paque PLUS(Fig 3)Fig 3.Blood sampleFicoll-Paque PLUSImportant:When layering the sample do not mix Ficoll-Paque PLUS and the diluted blood sample.4.Centrifuge at 400 g for 30-40 minutes at 1820 C.5.Draw off the upper layer using a clean Pasteur pipette,leaving the lymp-hocyte layer undisturbed at the interface(Fig 4,5).Care should be taken not to disturb the lymphocyte layer.The upper layer of plasma,which is essentially free of cells,may be saved for later use.p.9Procedure for washing the lymphocytes to remove platelets.1.Using a clean Pasteur pipette transfer the lymphocyte layer to a clean centrifuge tube.It is critical to remove all of the interface but a minimum amount of Ficoll-Paque PLUS and supernatant.Removing excess Ficoll-Paque PLUS causes granulocyte contamination,removing excess super-natant results in unnecessary contamination by platelets and plasma proteins.2.Add at least 3 volumes(6 ml)of balanced salt solution to the lymphocytes in the test tube.3.Suspend the cells by gently drawing them in and out of a Pasteur pipette.4.Centrifuge at 60100 g for 10 minutes at 1820 C.5.Remove the supernatant.6.Suspend the lymphocytes in 68 ml balanced salt solution by gently dra-wing them in and out of the Pasteur pipette.Fig 4.Lymphocytes Monocytes PlateletsGranulocytes ErythrocytesFig 5.Lymphocytes Upper layer removedPlasmaFicoll-Paque PLUS p.10 7.Centrifuge at 60100 g for 10 minutes at 1820 C.8.Remove the supernatant.9.The lymphocytes should now be suspended in the medium appropriate to the application.11.Expected ValueTypical results from our laboratory.Lymphocytes 95 5%of cells present in fraction are mononucleocytes,95 5%viability1,60 20%recovery of lymphocytes from the original blood2 sampleOther cells 3 2%granulocytes3,5 2%erythrocytes,0.5%of total platelets in the original blood sample remain1 Lymphocyte viability was determined by the Trypan blue exclusion test(13).2 The white blood cell count on the starting blood sample was done in a hemacytometer(14).A differential count of the white blood cells was then performed to determine the amount of agranulocytes in the starting blood sample.3 The differential cell count was obtained from a smear of the lymphocyte fraction treated with Wright s Stain(14).12.Factors Aff ecting Lymphocyte Isolation by the Ficoll-Paque PLUS ProcedureThe blood volume and tube diameter are factors determining the height of the blood sample in the tube and,consequently,the centrifugation time.Increasing the height of the blood sample in the tube increases red cell contamination.The separation,however,is not appreciably affected by the diameter of the tube.p.11Hence,a larger volume can be separated in a tube of larger diameter,chosen so that the height of the blood sample in the tube and the separation time are constant.The yield and the degree of purity of the lymphocytes depend on the efficiency of red cell removal.When erythrocytes in whole blood are aggregated,some lymphocytes are trapped in the clumps and,therefore,sediment with the erythrocytes.This tendency is reduced by diluting the blood.Dilution gives a better lymphocyte yield and reduces the size of the red cell clumps.Aggregation of erythrocytes is,however,enhanced at higher temperatures(37 C)which decreases yield,but at low temperatures(4 C)the rate of aggregation is decreased,increasing the time of separation.A temperature of 18-20 C gives optimum results.If problems are encountered when removing platelets from the lymphocyte fraction by the washing procedure,a second centrifugation in a 420%sucrose gradient layered over Ficoll-Paque PLUS will effectively remove the platelet contamination(12).The platelets will remain at the top of the sucrose gradient,and the lymphocytes will sediment through the sucrose gradient to the top of the Ficoll-Paque PLUS layer.13.How SuppliedFicoll-Paque PLUS is supplied as a sterile solution in bottles of 100 ml and 500 ml.14.Ordering InformationProduct Pack size Code no.Ficoll-Paque PLUS 6 100 ml 17-1440-02Ficoll-Paque PLUS 6 500 ml 17-1440-03Related productsFicoll-Paque PREMIUM 6 100 ml 17-5442-02Ficoll-Paque PREMIUM 6 500 ml 17-5442-03 p.12 15.References 1.Skoog,W.A.and Beck,W.S.,”Studies in the fi brinogen,dextran,and phyto-hemag glutinin methods of isolating leukocytes.”Blood 11:436454,1956.2.Byum,A.,”Separation of white blood cells.”Nature 204:793794,1964.3.Byum,A.,”Isolation of mononuclear cells and granulocytes from human blood.”Scand.J.Clin.Lab.Invest.21 Suppl.97(Paper IV):7789,1968.4.Harris,R.and Ukaejiofo,E.O.,”Tissue typing using a routine one-step lymphocyte separation technique.”Brit.J.Haematol.18:229235,1970.5.Thorsby,E.and Bratlie,A.,”A rapid method for preparation of pure lymphocyte suspensions.”Histocompatability Testing ed.P.I.Terasaki:665666,1970.6.Ting,A.and Morris,P.J.,”A technique for lymphocyte preparation from sto-red heparinized blood.”Vox Sang 20:561563,1971.7.Thorsby,E.,”Cell specifi c and common antigens on human granulocytes and lymphocytes demonstrated with cytotoxic heteroantibodies.”Vox Sang 13:194206,1967.8.Bain,B.and Pshyk,K.,”Enhanced reactivity in mixed leukocyte cultures after separation of mononuclear cell on Ficoll-Hypaque.”Transplantation Proceedings 4:163164,1972.9.Wottawa,A.,Klein,G.and Altman,H.,”A method for the isolation of human and animal lymphocytes with Ficoll-Urografi n.”Wiener Klin.Wochenschr.86:161163,1974.10.Wybran,J.,Chantler,S.and Fudenberg,H.H.,”Response to phytohe-magglutinin.”J.Immunol.110:11571160,1973.p.1311.Fotino,M.,Merson,E.J.and Allen,F.H.,”Instant lymphocytes”.Vox Sang 21:469470,1971.12.Peper,R.J.,Tina,W.Z.and Mickelson,M.M.,”Purifi cation of lymphocytes and platelets by gradient centrifugation.”J.Lab.and Clin.Med.72:842848,1968.13.Phillips,H.J.,”Dye exclusion tests for cell viability.”Tissue Culture:Methods and Applications ed.by Kruse,P.F.,Jr.and Patterson,M.J.,Jr.:406408(Chapter 3),Academic Press,1973.14.Brown,L.,Hematology:Principles and Procedures publ.by Lea and Febinger,Phila.,1973.p.14 p.15GE,imagination at work and GE monogram are trademarks of General Electric Company.Ficoll and Ficoll-Paque are trademarks of GE Healthcare companies.All third party trademarks are the property of their respective owners.2007 General Electric Company All rights reserved.First published March 1998All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them.A copy of these terms and conditions is available on request.Contact your local GE Healthcare representative for the most current information.GE Healthcare Europe GmbHMunzinger Strasse 5D-79111 FreiburgGermany GE Healthcare UK LimitedAmersham PlaceLittle ChalfontBuckinghamshire,HP7 9NA UKGE Healthcare Bio-Sciences Corp.800 Centennial AvenueP.O.Box 1327Piscataway,NJ 08855-1327USAGE Healthcare Bio-Sciences KKSanken Bldg.3-25-1Hyakunincho Shinjuku-ku,Tokyo 169-0073J Healthcare Bio-Sciences ABBjrkgatan 30751 84 UppsalaSweden71-7167-00 AG 02/2007 Elanders stervla 2007imagination at work