This document outlines three protocols for DNA fragmentation assays to analyze apoptosis. Protocol I uses Triton X-100 lysis buffer to isolate DNA from cells incubated with effectors. The DNA is extracted, precipitated, and run on an agarose gel. Protocol II uses SDS lysis buffer instead of Triton X-100. Protocol III labels cells with radioactive thymidine overnight before using them in a lytic assay to measure radioactive thymidine release, indicating DNA fragmentation.
This document outlines three protocols for DNA fragmentation assays to analyze apoptosis. Protocol I uses Triton X-100 lysis buffer to isolate DNA from cells incubated with effectors. The DNA is extracted, precipitated, and run on an agarose gel. Protocol II uses SDS lysis buffer instead of Triton X-100. Protocol III labels cells with radioactive thymidine overnight before using them in a lytic assay to measure radioactive thymidine release, indicating DNA fragmentation.
This document outlines three protocols for DNA fragmentation assays to analyze apoptosis. Protocol I uses Triton X-100 lysis buffer to isolate DNA from cells incubated with effectors. The DNA is extracted, precipitated, and run on an agarose gel. Protocol II uses SDS lysis buffer instead of Triton X-100. Protocol III labels cells with radioactive thymidine overnight before using them in a lytic assay to measure radioactive thymidine release, indicating DNA fragmentation.
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$%