Preparingacellblockfromcelllines/cellsUSPensions.

Celllinesorpurifiedcellsuspensionsareavaluabletoolto

provideknownpositivecontrols

providehomogeneoustissueinunlimitedamount,characterizablebymolecular,cytogeneticandbiochemicalmeans

providemodelsforex-vivocellcycleandactivation-dependenteffects

Cellsgrownincultureorobtainedex-vivocanbeeasilycytospunorsmearedonaslide.Howeverconditionsarequitedifferentfromatissueblock,fixedandembeddedinparaffin.Tothisendcellsuspensionscanbefixed,embeddedinagar(aninertmaterial),andprocessedasapieceoftissue.

Livecellpreparation.Prepare1%Agarose,cellculturegrade,inisoosmoticPBS.YouneedtoboiltodissolveitinPBS.Checkforlossofwaterasvaporduringboilingandreplacewithdistilledwater.Bringtoapprox50C°.Theagarsolidifybelowthispoint.Prepareyourcellsasacellsuspensioninminimalamountofmedium,atroomtemperature.Tomakea0.5mlagarblockyoumayneedbetween10to20x10⁶cells.Youcanscalethisdowntoaslittleas0.5x10⁶in50µlbutyourcellswillbeverysparseonsection.Prepareinadvance1.5mlEppendorftubes(orsmallPCRtubesformicro-preparations)towhichyoucutoffanddiscardedtheconicalbottom(!).Capthetube.Mixevenlyandthoroughlyyourcellswith0.5mlofagar(orless)andveryquicklytransfertothecappedinvertedtube.Nobubbles!!YoumaywantalsotocutthePipettetip,soyouhavealargeropening.Placethestillmoltenagarandtubeoniceuntilissolid.Thenopencarefullythecap,andgentlyextracttheagarcylinderfromitsbase(thecap).Atthispointyoucancuttheagarpieceintwowitharazorblade,freezehalfandfixinformalintheother.Yourcellsarestillaliveandbiochemicallyactiveatthispoint.

Fixedcellpreparation.Proceedasabove,butyoufixthecellsbefore.ThenyoucanuseAgaroseinPBS,regardlessofosmolarity.Fixingthecellsbeforeembedding,preventsmovementoflABIleantigensorlossofshort-livedmolecules.Findhereanexampleofacellblockpreparation.

Tricks.Itisveryconvenienttohaveonthesameblockapositiveandanegativecontrol,buthowtodistinguishtwosemi-transparentblockofagarandidentical-lookingcells?PutatinyamountofIndiaInk(asyourSurgicalPathologist)inthemoltenagarbeforeembeddingthecells.Mustbenotmorethanafaintgreyhue,enoughtodistinguishtheblockwithnakedeyeandatthemicroscope.

YoumayalsowanttocheckasimilarcellblocktechniqueattheNIHwebsite.

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。