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27845 Irma Lee Circle, Unit 101, Lake Forest, Illinois 60045

Spherotech, Inc.

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SPHEROTM Technical Note

STN-7 Rev B 080907

Separation of Mononuclear Cells from Peripheral Blood using SPHEROTM Goat anti-Mouse IgG Magnetic Particles

MATERIALS: 1.Gtanti-MsIgGMagneticParticles,1%w/v,Cat.#MM-40-10orMMXA-40-10,~2x108particles/mL 2.Appropriatemonoclonalantibody(anti-CD3,CD4orCD8etc.) 3.AppropriateFITCconjugates 4.DulbeccoPBS 5.Fetalbovineserum 6.Histopaque 7.Paraformaldehydefixative 8.FlexiMagSeparator,Jr.,Cat.#FMJ-1000 PROCEDURES: SEPARATION OF MONONUCLEAR CELLS FROM PERIPHERAL BLOOD Collect peripheral blood by venipuncture of the antecubital vein. Draw blood into heparin Vacutainer tubes, transferto50mLpolypropylenecentrifugetubesanddilutewithanequalvolumeofcalciumandmagnesium-free Dulbecco'sphosphatebufferedsaline. Layer10to20mLaliquotesofdilutedbloodontoanequalvolumeofHistopaquein50mLtubes.Centrifugefor 30minutesatambienttemperatureusingacentrifugationforceof400gattheblood/Histopaqueinterface.Aspirate thelymphocytebandintoa15mLcentrifugetubeandbringthevolumeto14mLwithDulbeccoPBScontaining 2%v/vheat-inactivatedfetalbovineserum,or5%plasmaproteinfraction.Pelletthecellsbycentrifugationat4oC for7minutesat450g,washwith14mLofPBS,andrecentrifuge.Resuspendthefinalcellpelletin1-2mLbuffer, andaviablecellcountperformed.Onlycellpreparationswithaviabilityof>95%shouldbeused. COATING OF ANTI-MOUSE IgG MAGNETIC PARTICLES WITH MONOCLONAL ANTIBODY Add2to5µgofmonoclonalantibodyto100µLof1%w/vGtanti-MouseIgGcoatedmagneticparticles(~2x107 particles)in12x75tubes.Incubatethemixturewithoccasionalmixing,at4oCforatleast30minutes.Washthe particlesthreetimeswith2mLofDulbeccoPBS. INCUBATION OF MAGNETIC PARTICLES AND CELLS Pipet ~2x106 cells into 12x75 mm glass test tubes and add monoclonal antibody coated magnetic particles to thetubesattheparticletocellratioof5~20to1.Incubatethetubesforatleast30minutesat4oContherotator ataspeedsettingof2to4rpm.SeparatethemagneticparticleswiththeFlexiMagSeparatorJr..Transferthe supernatantliquid,containingtheunboundcells,toa12x75mmtubeandcountthecellsinahemacytometer, and/oranalyzebyimmunofluorescence.

Tel.: 800-368-0822 or 847-680-8922; Fax: 847-680-8927; E-Mail: [email protected] Visit us on the web at http://www.spherotech.com

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27845 Irma Lee Circle, Unit 101, Lake Forest, Illinois 60045

Spherotech, Inc.

DETECTION OF CELL DEPLETION BY IMMUNOFLUORESCENCE Determine the proportion of target cells in the mononuclear cell suspension pre- and post-separation by immunofluorescencestainingandexaminevisuallyorbyflowcytometry. Incubate the remaining cells (unbound cells) after immunomagnetic separation with 40 µL of anti-target cell monoclonalantibodyforatleast30minutesat4oCwithoccasionalmixing.Washthecellsthreetimeswith2mL ofthebuffer.Resuspendthecellpelletwith50µLofa1/10dilutionofFITC-conjugatedanti-MouseIgGantibody inPBS/FBSbuffer.Incubateandwashthecellsasdescribedpreviously.Resuspendthefinalcellpelletin300 µLofparaformaldehydefixativeandstoreat4oCinthedarkuntilanalyzedontheflowcytometerorfluorescence microscope. Performthevisualdetectionusingafluorescencemicroscope.Examinethecellsundernormalilluminationand countthenumberofcells.Re-examinethesamefieldunderepi-illumination,andcountthenumberoffluorescent cells.Categorizeatleast200cellsineachpreparation. For flow cytometer evaluation, calibrate the flow cytometer and establish the lymphocyte gate based on the forward/sidescatterprofiles,orbyback-gatingaccordingtothemanufacturer'sdirections.Classifyatleastten thousandcellsfromeachtubeandanalyzethedatausingappropriatesoftware. Controlsshouldconsistofmononuclearcellsincubatedwith(i)monoclonalantibodiestoobtainpre-depletion targetcellpercentages;(ii)particlescoatedwiththeisotypecontrolmonoclonaltodeterminenon-specificdepletion ofthetargetcellsbytheparticles;(iii)eachofthepost-depletionanalysisreagentsindependentlytoevaluate backgroundbindingofthesereagents;(iv)theLeucogatereagentforcytometricanalysistodeterminethepurity ofthemononuclearcellpreparationandtheaccuracyofthegatingprocedure. Calculatethepercentdepletionas: %Targetcellspost-depletion/%Targetcellspre-depletionx100 NOTE:Forcellseparationusing1~2ummagneticparticlesat0.25%w/v,suchasMMXA-10-10orMMX-1010,usethesamevolumeasinMMXA-40-10orMM-40-10at1%w/v.

Tel.: 800-368-0822 or 847-680-8922; Fax: 847-680-8927; E-Mail: [email protected] Visit us on the web at http://www.spherotech.com

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