DNA Fragmentation Assays for Apoptosis Protocol

Protocol I: Triton X-100 Lysis Buffer

In 96 flat-wells plate, incubate 4x10 6 target cells 40 wells of 10! per well" wit# $esire$
concentration of effectors 10! target cells per well"% &fter incubation, collect t#e cell
sa'ple in 1%! 'l eppen$orf tube, spin $own, resuspen$ wit# 0%! 'l (B) in 1%! 'l
eppen$orf tubes, an$ a$$ !!ul of lysis buffer for *0 'in on ice 4o+"% +entrifuge t#e
eppen$orf tubes in col$ at 1*,000 g for ,0 'inutes% Transfer t#e sa'ples to new 1%! 'l
eppen$orf tubes an$ t#en extract t#e supernatant wit# 1-1 'ixture of p#enol-c#lorofor'
gentle agitation for ! 'in followe$ by centrifugation" an$ precipitate in two e.ui/alence
of col$ et#anol an$ one-tent# e.ui/alence of so$iu' acetate% )pin $own, $ecant, an$
resuspen$ t#e precipitates in ,0ul of $eioni0e$ water-12ase solution 0%4'l water 3 !ul
of 12ase" an$ !ul of loa$ing buffer for ,0 'inutes at ,4o+% &lso insert *ul of 5in$i III
'ar6er 1*ul of )toc6 I7" on t#e outer lanes% 1un t#e 1%*8 gel at !7 for !'in before
increasing to 1007%
(rotocol II- )9) LysisBuffer
&$$ )9) lysis buffer to t#e incubate$ cell sa'ples prepare$ as in (rotocol I"%
Stock I:Triton X-100 Lysis Buffer 40 'l of 0%! : ;9T& ! 'l of 1 : Tris+l buffer p5
<%0 ! 'l of 1008 Triton X-100 !0 'l of 5*=
Stock II: )9) Lysis Buffer
Stock III: 1%*8 &garose >el
(repare a stoc6 of * liter of 1X T&; i%e%, * liter 3 40'l of !X T&;"% &$$ *%4g of agarose
power1%*8 agarose" to *00'l of 1X T&; solution an$ 'icrowa/e for 4 'in at #ig#
power% T#en cool t#e gel to !0o+ an$ a$$ *!ul of et#iu' bro'i$e before pouring it into
t#e gel plate% Insert co'b an$ let t#e gel poly'eri0e$%
Stock IV: 5in$i III :ar6er !0 ?b la'$a 92&" 4ul of 5in$i III :ar6er 16ul of
9eioni0e$ @ater 4ul of Loa$ing Buffer
Protocol II: 92& Arag'entation &ssay /ia 9ip#eyla'ine
In *4-wells plate, incubate ! X 106 targets wit# $esire$ nu'ber of effectors% &fter
incubation, transfer t#e sa'ples to 1!'l tubes, centrifuge for ,0 s at 1!00g, an$
resuspen$ in !'l of lysis buffer )toc6 I7" for 1! 'in on ice% +entrifuge t#e sa'ples for
*0 'in at *4,000g to separate #ig#-'olecular-weig#t c#ro'atin fro' clea/age pro$ucts%
1esuspen$ t#e pellet in ! 'l of buffer stoc6 7"% Treat t#e supernatants an$ pellets wit#
t#e $ip#enyla'ine reagent )toc6 7I" an$ incubate at ,40+ for 16-*4 #r before
colori'etric assess'ent%
Stock IV: Lysis buffer at p5 <%0 !': Tris-5+l *0': ;9T& 0%!8 Triton X-100
Stock V: Buffer at p5 <%0 10': Tris-5+l 1': ;9T&
Stock VI: 9ip#enyla'ine reagent lig#t sensiti/e" 1%!g of $ip#enyla'ine stea'-$istille$"
100'l acetic aci$ re$istille$" 1%!'l of conc% sulfuric aci$
=n t#e $ay of usage, a$$ 0%10'l of ag acetal$e#y$e 16'gB'l" to *0'l of t#e
$ip#enyla'ine reagent%
Protocol III: 92& Arag'entation /ia ,5-T$1
! X 106 target cells were labele$ wit# !0Cl of ,5-T$1 1 '+iB'l" o/ernig#t in 10 'l of
'e$ia% T#e next $ay, t#e cells were was#e$ ,X wit# 10'l of (B) an$ incubate$ in 10'l
of 'e$ia to c#ase out unincorporate$ cytoplas'ic ,5-T$1% &fter incubating for * #rs, t#e
cells were was#e$ ,X wit# (B) an$ t#en use$ in lytic assay un$er t#e sa'e con$itions as
t#e !1+r release assay in 96 /-well plates% &t t#e en$ of t#e assay, eac# well was treate$
wit# *0Cl of 1%08 Triton-X on ice for ! 'inutes, followe$ by centrifugation at 1!00g in a
Bec6'an T-D6 rotor for 1! 'inutes% 100Cl of t#e supernatant were #ar/este$ fro' eac#
well an$ counte$ in a scintillation counter% Total count was obtaine$ by resuspen$ing t#e
cells prior to #ar/esting, an$ a$$ing 0%18 )9) to solublili0e t#e cells% T#e 8 ,5 release$
was calculate$ wit# an e.uation analogous to t#at for 8!1+r release$%