TUNEL labelling of HeLa cells (Modified from Kirstins protocol) Date: Cells: Treatment: Comments:

1. Induce apoptosis of 2 x 105 HeLa cells in suspension. i.e. trypsini e HeLa cells (it is important t!at t!e cells are in sin"le cell suspension and not clumpy #efore you add apoptosis inducer). Let cells reco$er at %&o' in complete media #efore addition of apoptosis inducer. 2. (fter apoptosis is completed) spin do*n cells in +,-*ell plate. (1500 rpm for % min). %. .s cells in 200 ul ice cold acidic met!anol (5/ acetic acid0 +5/ met!anol1 -20o'). Lea$e plate o$erni"!t at 2o'. 2. 3pin do*n cells (1400 rpm for % min). 5. .s cells in 200 ul 563 7 0.1/ 8*een-20. 9ently pipet up and do*n to #rea: up any cell clumps. ,. 3pin do*n cells (1400 rpm for % min). &. .s cells in 20 ul of 8;<=L mix. -8;<=L mix is 1+ ul of 8;<=L la#el 1 ul of 8;<=L en yme

-3et up premix for >>>>>> *ells.

-incu#ate for 1-2 ! at %&o'. (2 ! is #etter)

4. 8op up cells *it! 563 72/ ?'3 and spin do*n. @as! *it! 563 7 2/ ?'3 and t!en finally) resuspend in 563 7 2/ ?'3. +. .ead on ?('3 at ?L1 for "reen 8;<=L. (lternati$ely) 8;<=L assay can #e done on co$erslips and analy ed #y microscopy. In t!is case) HeLa cells are plated on co$erslips (approx. 1 x 105 cells per co$erslip)) apoptosis is induced) cells are fixed for 15 min. *it! ice cold acidic met!anol) permea#ili ed as in step 5 and t!en incu#ated *it! t!e 8;<=L mix. @as! 2 x *it! 563 and t!en mount co$erslips *it! 5rolon". 9reen 8;<=L is analy ed at 244 nm and red 8;<=L is analy ed at 52% nm.