Celllinesorpurifiedcellsuspensionsareavaluabletoolto 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.Preparingacellblockfromcelllines/cellsUSPensions.
Cellsgrownincultureorobtainedex-vivocanbeeasilycytospunorsmearedonaslide.Howeverconditionsarequitedifferentfromatissueblock,fixedandembeddedinparaffin.Tothisendcellsuspensionscanbefixed,embeddedinagar(aninertmaterial),andprocessedasapieceoftissue.
