Cellnumber,speedandtrivialdetailsaffectcytospin.

Basicprotocol:

PrepareacellsUSPensionofnotmorethan0.5x10⁶cells/mlofprotein-containingmedium.Pre-labeltheslides.

Procedureforoldtypecytocentrifuge(becareful,thisisnotbiohazardproof!!)

Preparetheslidesmountedwiththepaperpadandthecuvetteinthemetalholder.

Loadupto200µlofthissuspensionineachcuvette.Spinat800rpmfor3min.Extracttheslide,paperandcuvettewithoutdisarranging.Carefullydetachthecuvetteandthepaperwithoutdamagingthefreshcytospin.Isveryimportanttoholdfirmlytogetherglassslideandcuvettewhenextractingfrommetalholder.MarktheareaaroundthecytocentrifugedcellswithdrypointorpermanentMarker.Proceedwitheitherimmediatefixationordrying.Storeunfixedcytospinsformax2daysatroomtemperature.

Ifyouhavelittlecells:Asfewas5x10⁴cellscanbecytospun.

Proceedasfollows:Preparethecellsuspensionwith10%FCS.Preruntheloadedcytocentrifugewith50µlmedium+serum,inordertowetthepaperpads.

Runyoursamplewithnotfewerthan100µlandnotmorethan250µl.

Providerofcytospinmaterial:

ShandonInc.,171IndustryDrive,Pittsburgh,PA15275.

A:ShandoncardsCatn°190005B:re-usedcuvettewithgluedpaperresiduesremoved.C:Shandonpre-madecuvettesCatn°5991040(boxof50).