TUNEL and nuclear staining for FACS analysis

The aim of this staining is to combine TUNEL staining with immunostaining of nuclear/membrane/cytoplasmic antigens and nuclear dye (7-AAD).
You should pre-test your antibodies for performance with the fixation method for TUNEL.

Positive control.

* Exponentially growing Ramos cells are incubated for 4hrs. with 18 µM Camptothecin (CPT; Fisher/ Acros Cat. 27672-1000, FW 348.35. Dissolve in DMSO)  or vector alone. Viability should be in both >90%. You should expect 3x to 10x times apoptotic cells (AnnexinV+, 7AAD-) in the CPT-treated sample. Aliquots of induced and uninduced Ramos can be kept at +4°C in 70% EtOH.

TUNEL staining.

1- Resuspend the cells in complete medium at a convenient concentration (You may use tubes or 96 U-bottom microtiter wells. Each microtiter well contains 300 µl and you may not stain less than 500.000 cells per well).

2- Place 1 to 3 millions cell per well / tube.
3- Fix in BD erythrocyte lysing solution (Becton Dickinson Cat. n° 349202 Stock is 10x. Dilute in Water) for 10 min at RT.
4- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added (TBS-T).
5- Fix for minimun 10 min. in chilled 70% Ethanol. EtOH volume shuld be at least 5x the cell volume. In this step is critical  to avoid proteins in the medium, because they coagulate and make a cloud of dust. Cells also tendo to aggregate. Best would be pouring the cell suspension in the EtOH.

At this point the cells can be stored at +4C°.

6- Wash twice the cell suspension in TdT reaction buffer without cobalt chloride( 30 mM Trizma base, pH 7.2, 140 mM sodium cacodylate).
7- Warm up the tubes or the plate and the TdT reaction buffer to 37C°.
8- Thaw one of each of the TUNEL reaction tubes and set up an aliquot without TdT (this will be your negative control). (In Situ Cell Death Detection Kit, Fluorescein, Boehringer Mannhein/Roche, Cat 1684795. 50 tests). Mix the TdT and the nucleotide solution. Dilute both  the negative and the positive reaction solution 1:3 with cobalt chloride-free, warm TdT reaction buffer. Diluting the TUNEL solution lowers the background by lowering the cobalt content and makes more clean the reaction.
9- Allow the reaction to proceed for 30 min at 37C°.
10- Wash once with cold staining medium and proceed with the non-specific blocking step.
11- Incubate with the primary antibodies or mixtures of primary antibodies from 1 hr to O/N.
12- Wash twice with staining medium.
13- Incubate with the secondary antibodies for 1 hr. If you use biotin-conjugated antibodies, repeat the washing after the secondary and then incubate with the conjugated Avidin for the same amount of time.
14- After a final two washes, resuspend the cells in PBS, BSA, formalin, in approx 300-400 µl for analysis.

If you like to do DNA staining with 7AAD, you may use 7AAD at 25µg/ml and incubate for at least 30 min. Be aware that 7AAD staining combined with other fluorochromes is very cumbersome because of compensation problems.