Cell cycle analysis Propidium iodide staining Experiment: Aim: Samples: Sample Cell count Vol req Sample

Date:

Cell count

Vol req

Procedure: 1. Transfer 1.5X1 ! cells into a conical tu"e. #. C$ill on ice for 5%. &. Spin: 5 '( 5%( )T. Sa*e pellet. +. ,oosen pellet. Add 1ml ice cold 1XP-S. 5. Spin: 5 '( 5%( )T. Sa*e pellet. !. ,oosen pellet. )esuspend in 1ml ice cold 1XP-S. .. Add #ml ice cold 1 / met$anol to samples 0$ile *ortexin'. 1. 2ncu"ate: 2ce( & %. Samples can "e stored $ere for a 0ee3. 4. Spin: 5 '( 5%( )T. Sa*e pellet. 1 . ,oosen pellet. 11. Add 5 ul propidium iodide solution in 1XP-S. 5inal concentration of P2 6 #5u'7ml. Vortex samples. 1#. Add 5 ul )8ase in 1X P-S to samples. 5inal concentration of )8ase6 1 u'7ml. Vortex samples. 1&. 2ncu"ate: & %( )T( dar3. 1+. Anal9se "9 5lo0 c9tometr9 0it$in ! %. )un lo0 and collect # ( e*ents.

15. 2f usin' 5ACS cali"er: :SE 5,& ; Dot plot( 5SC and SSC( X axis *7s < axis. ; Dot plot( 5,&;0 and 5,&;a( X axis *7s < axis. T$is is to differentiate "et0een cells in cell c9cle *7s dou"lets. =ate for cell c9cle population. Cells ri'$t on top are aneuploid( to t$e ri'$t are dou"lets. ; >isto'ram from 'ated population in Dot plot #( 5,&;a and counts( X axis *7s < axis. T$is s$o0s apoptotic( = 7=1( S and =#7? populations. Ad@ust 5,& *olta'e to 'et = 7=1 around 5 7# and =#? dou"le of t$at A 1 7+ . ; :se ma'nifier to Boom on cell c9cle population 'ate in dot plot #. Can t$en ma3e more 'ates to see num"er of cells in eac$ p$ase of t$e cell c9cle.