Identification of apoptotic cells based on the presence of DNA strand breaks

DNA fragmentation during apoptosis generates a large number of DNA strand breaks. The 3'-OH termini in the strand breaks can be detected by attaching a fluorochrome to them. This is generally done by using, directly or indirectly (e.g. via biotin or digoxygenin), fluorochrome-labelled deoxynucleo-tides in a reaction preferentially catalysed by exogenous TdT, as originally described in refs 14 and 16 and discussed in Chapter 2. Of all the markers of DNA strand breaks, BrdUTP appears to be the most advantageous with respect to sensitivity, low cost, and simplicity of the reaction (17). This deoxynucleotide, once incorporated into DNA strand breaks, is detected by an FITC-conjugated anti-BrdU antibody. It should be stressed that detection of DNA strand breaks requires cell pre-fixation with a cross-linking agent such as formaldehyde, which, unlike ethanol, prevents the extraction of small sections of the fragmented DNA. Thus, despite cell permeabilization (with ethanol) and the subsequent cell washings during the procedure, the DNA content of early apoptotic cells (and with it the number of DNA strand breaks) is not markedly diminished compared with unfixed cells.

Protocol 5. Detection of DNA strand breaks

Reagents

• prepare fixatives—1st fixative: 1% methanol-free formaldehyde (available from Polysciences Inc., Warrington, PA) in PBS, pH 7.4; 2nd fixative: 70% ethanol

• the TdT reaction buffer (5 x) contains: potassium (or sodium) cacodylate, 1 M; Tris-HCI, 125 mM, pH 6.6; bovine serum albumin (BSA), 1.25 mg/ml

The buffer, TdT, and CoCI2 are available from Boehringer Mannheim, Indianapolis, IN.

• BrdUTP stock solution: BrdUTP (Sigma) 2 mM (100 nmoles in 50 ^1) in 50 mM Tris-HCI, pH 7.5

• FITC-conjugated anti-BrdU mAb solution (per 100 n.l of PBS): 0.3 (ig of anti-BrdU FITC-conjugated mAb (available from Becton Dickinson); 0.3% Triton X-100; 1% BSA

• rinsing buffer. Dissolve in PBS: Triton X-100, 0.1% (v/v); BSA, 5 mg/ml

• PI staining buffer. Dissolve in PBS: PI, 5 |j,g/ml; DNase-free RNase A, 200 ng/ml

Method

1. Fix cells in suspension in 1% formaldehyde for 15 min on ice.

2. Centrifuge (300 g, 5 min), resuspend the cell pellet in 5 ml PBS, centrifuge (300 g, 5 min), resuspend the cells (approximately 106 cells) in 0.5 ml of PBS.

3. Add the above 0.5 ml aliquot of cell suspension into 5 ml of ice-cold 70% ethanol. The cells can be stored in ethanol, at -20°C for several weeks.

4. Centrifuge (200 g, 3 min), remove ethanol, resuspend cells in 5 ml PBS, centrifuge (300 g, 5 min).

5. Resuspend the pellet (not more than 106 cells) in 50 ¡ulI of a solution which contains:

• 5 jxl of CoCI2 solution

6. Incubate cells in this solution for 40 min at 37°C (alternatively, incubation can be carried at 22-24°C overnight).

7. Add 1.5 ml of the rinsing buffer, centrifuge (300 g, 5 min).

8. Resuspend cells in 100 |xl of FITC-conjugated anti-BrdU mAb solution.

9. Incubate at room temperature for 1 h or at 4°C overnight. Add 2 ml of rinsing buffer, centrifuge (300 g, 5 min).

10. Resuspend the cell pellet in 1 ml of PI staining solution containing RNase.

11. Incubate for 30 min at room temperature in the dark.

12. Analyse cells by flow cytometry.

• illuminate with blue light (488 nm laser line or BG12 excitation filter)

• measure green fluorescence of FITC-anti-BrdU mAb at 530±20 nm

• measure red fluorescence of PI at >600 nm

Commercial kits

Phoenix Flow Systems and PharMingen Inc., (San Diego, CA, USA) provide kits to identify apoptotic cells based either on a single-step procedure utilizing TdT and FITC-conjugated dUTP (APO-DIRECT™) or TdT and BrdUTP, as described above (APO-BRDU™). A description of the method, which is nearly identical to the above, is included with the kit. Another kit (ApopTag™), based on two-step DNA strand break labelling with digoxygenin-16-dUTP by TdT, is provided by ONCOR Inc., (Gaithersburg, MD, USA).

Analysis of DNA strand breaks by laser-scanning cytometry (LSC)

1. Add 300 m-I of cell suspension in tissue culture medium (with serum) containing approximately 20000 cells into a cytospin chamber. Cyto-centrifuge at 1000 r.p.m. (—110 g) for 6 min.

2. Without allowing the cytospins to dry completely, pre-fix cells in 1% formaldehyde in PBS for 15 min on ice.

3. Transfer the slides to 70% ethanol and fix for at least 1 h; the cells can be stored in ethanol for several days.

4-9. Follow steps 4-9 above, as described for flow cytometry. Small volumes (50-100 jxl) of the respective buffers, rinses, or staining solutions are carefully layered on the cytospin area of the slides held horizontally. At appropriate times these solutions are removed with a Pasteur pipette (or vacuum suction pipette). Small pieces (2.5 cm x 2.5 cm) of thin polyethylene foil may be layered on slides atop the drops to prevent drying. The incubations should be carried out in a moist atmosphere to prevent drying at any step of the reaction.

Zbigniew Darzynkiewicz et al. Protocol 5. Continued

10. Rinse the slide in PBS and mount the cells under a coverslip in a drop of the PI staining solution containing RNase A. If the preparations are to be stored for a longer period of time (hours, days, at 4°C), the coverslips are mounted in a drop of a mixture of glycerol and PI staining solution (9:1).

11. Measure cell fluorescence on LSC.

• excite fluorescence with 488 nm laser line

• measure green fluorescence of FITC-anti-BrdU mAb at 530±20 nm

• measure red fluorescence of PI at >600 nm

Identification of apoptotic cells is based on their intense labelling with FITC-anti-BrdU mAb, which frequently requires use of an exponential scale (logarithmic photomultipliers) for data acquisition and display (Figure 6). Simultaneous measurement of DNA content makes it possible to identify the cell cycle position of both cells in apoptotic and non-apoptotic populations.

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Figure6. DNA strand break labelling in apoptotic cells. To induce apoptosis HL60 cells were treated with CPT for 3 h as described (16,17). DNA strand breaks were labelled with BrdUTP and cellular DNA was counterstained with PI, as described in Protocol 5. Cellular fluorescence was measured by flow cytometry (FACScan). Note that, preferentially, S phase cells are undergoing apoptosis.

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DNA Content

Figure6. DNA strand break labelling in apoptotic cells. To induce apoptosis HL60 cells were treated with CPT for 3 h as described (16,17). DNA strand breaks were labelled with BrdUTP and cellular DNA was counterstained with PI, as described in Protocol 5. Cellular fluorescence was measured by flow cytometry (FACScan). Note that, preferentially, S phase cells are undergoing apoptosis.

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