PRINS Characterization of Micronuclei

The advantages related to the application of primed in situ labeling (PRINS) with respect to fluorescence in situ hybridization (FISH) are well known, and they are discussed in detail elsewhere in this book (see Chapters 1, 3, and 6). In particular, the background noise is lower with PRINS than with FISH, which is crucial in interphase analysis when the discrimination of true reaction signals from aspecific fluorescent spots is difficult. Accordingly, in the micronucleus assay, higher sensitivity and sensibility were found after PRINS than after FISH (5). A second important advantage of PRINS is that the protocol can be applied easily on cell types of different species, provided that knowledge of the target DNA sequence exists, because the labeling procedure is based on the use of oligonucleotide primers, which are available from many companies or institutional facilities. This aspect is very important because the optimization of commercially available probes for FISH is mainly restricted to diagnostic purposes and, therefore, to the human genome.

In this chapter the procedures for single- and dual-color PRINS detection of repetitive DNA sequences in micronuclei of mammalian cells are described. In addition, a guide to the interpretation of the results and to the choice of most appropriate labeling combinations is provided.

Micronuclei

Fig. 2. The detection of repetitive sequences in micronuclei allows one to infer the mechanism of origin: (A) the detection of centromeric sequences makes possible the distinction of centromere-carrying micronuclei, which derive from chromosome loss, from centromere-negative micronuclei representing acentric fragments. Agents acting with a prevalent clastogenic mechanism will produce a large majority of centromere-negative micronuclei; on the contrary, aneuploidogenic agents will specifically increase the frequency of centromere-positive micronuclei. (B) The dual-color detection of mouse centromeric and pericentromeric sequences (tandem labeling) allows one to distinguish among three classes of micronuclei, those positive for both sequences (representing whole chromosomes), those carrying the pericentromeric satellite only (representing huge chromosome fragments deriving from breakage at the pericentromeric heterochromatic region), and the "negative" ones which represent acentric fragments; (C) after dual-color detection of centromeric and telomeric sequences, four categories of micronuclei are expected, which correspond respectively to segregation errors before or during anaphase, to acentric chromosome fragments, to acentric chromatid fragments.

2. Materials

2.1. Sample Preparations

1. Fully equipped cytocentrifuge (Shandon "Cytospin," UK; see Note 1).

2. Hank's balanced salt solution without calcium and magnesium (an alternative: cell culture medium without serum).

3. Fixative solution (not required for slide preparation by cytocentrifuge): 3:1 mixture of methanol:glacial acetic acid, freshly prepared. Keep on ice after preparation (see Note 2).

5. Microscope slides (see Note 3).

6. Coplin jars with screwed cap.

7. Contrast phase microscope.

2.2. Single-Color Detection of Repetitive DNA Sequences in Micronuclei

1. Cell preparations, fixed on slides and maintained at -20°C.

2. Thermocycler equipped with in situ polymerase chain reaction (PCR) block (see Note 4).

3. The oligonucleotide corresponding to the target sequence. Synthesis of primers is conducted by several companies or facilities; oligos are stable for years at -20°C. Make aliquots to avoid repeated thawing/freezing.

4. Cocktail of dATP, dGTP, and dCTP, 5 mM each. Batch solutions of deoxy-nucleoside triphosphates must be appropriately diluted in pure (e.g., milliQ) H2O. Store 10- to 20-pL aliquots at -20°C. The cocktail is stable for at least 1 yr.

5. dTTP, 0.5 mM (appropriately diluted from the batch solution in pure H2O). Store in 10- to 20-pL aliquots at -20°C. Stable for at least 1 yr.

6. 1 mMDigoxigenin-11-dUTP; see Note 5), obtained from Roche Applied Science (Monza, Italy). Store at -20° C.

7. Taq DNA polymerase (Promega, storage buffer B, 5 U/pL; see Note 6).

8. Denaturing solution: 0.01 MNaOH, 1 MNaCl. Prepare freshly from the stock solutions (0.5 M NaOH, 2 M NaCl) and discard after use.

9. 0.01 M Tris-HCl, pH 7.6. Prepare from Tris-buffer, adjust pH, and autoclave. Store at 4°C and discard within 2 wk from the first utilization.

10. Stop solution: 50 mM NaCl, 50 mM ethylene diamine tetraacetic acid (EDTA). Prepare freshly from the stock solutions (2 M NaCl; 0.5 M EDTA, pH 8.0) and discard after use.

11. 20X standard saline citrate (SSC): 3 Msodium chloride, 0.3 Mtrisodium citrate. Store at room temperature.

12. 4X SSC, 0.1% Tween-20, pH 7.0. Dilute 100 mL of 20 X SSC in 380 mL of pure H2O, adjust pH, and bring to 500 mL. Add 500 pL Tween-20. Keep at 4°C and discard after 2 wk.

13. Phosphate-buffered saline (PBS): 140 mMNaCl, 2.7 mMKCl, 10 mMNa2HPO4, and 1.8 mMKH2PO4, pH 7.4. Autoclave and store at 4°C. Discard 2 wk after the first use.

14. PBST: PBS, 0.1% Tween-20. Store at 4°C and discard after 2 wk.

15. 10X blocking solution (Roche Applied Science). Prepare 2-mL aliquots and store at -20°C. Working aliquots can be kept at 4°C for several weeks. Discard if they become turbid.

16. Fluorescein-conjugated anti-digoxigenin mouse antibodies (Fab fragments; Roche Applied Science). Store at -20°C according to manufacturer instructions. To avoid repeated thawing/freezing, prepare 20-pL aliquots. The working aliquot can be kept for several weeks at 4°C and stored in the dark (see Note 5).

17. DAPI/antifade solution: 1.5 pg DAPI in 1 mL of Vectashield (Vector Laboratories, UK).

20. Coplin jars.

2.3. Dual-Color Detection of Repetitive DNA Sequences in Micronuclei

To the list of Subheading 2.2., add the following materials:

1. 1 mMBiotin-16-dUTP, obtained from Roche Applied Science. Store at -20°C.

2. Cy3-conjugated ExtrAvidin® (Sigma-Aldrich, Milan, Italy). Keep at 4°C according to the manufacturer's instructions, stored in the dark. Use a 1:10 working dilution for preparing the detection mix, which is a 1:300 final dilution.

3. Methods

3.1. Sample Preparations

This section describes the necessary steps to make slide preparations according to the requirements of the PRINS protocol. However the reader must refer to specific reviews on micronucleus assay (3,4,6) to define the correct experimental planning for cell treatment and harvesting (see Note 8). Slides can be prepared by hand from a cell suspension of previously fixed cells. The most efficient and recommended way to prepare slides is to use a cytocentrifuge (see Note 1).

1. A monocellular suspension must be used. When necessary, apply the appropriate digestion steps with proteolytic enzymes (see Note 9). Estimate the cell number, and then centrifuge the cell suspension. If a cytocentrifuge is available, go directly to step 5.

2. Discard the supernatant, and then resuspend the pellet in few drops of cool fixative solution (freshly prepared, 3:1 methanol:glacial acetic acid; see Note 2). After the pellet is completely resuspended, bring to 1.0 to 1.2 mL with fixative solution (the suspension should remain opalescent).

3. On perfectly cleaned slides (see Note 3) drop, from 5 to 10 cm of height, one to two drops of the cell suspension. Tilt immediately the slide at 45° angle, and wait for the complete fixative evaporation.

4. Check the quality of the preparation at the contrast phase microscope: cells must be evenly spaced and nonoverlapping; cytoplasm must be well preserved (see Note 10). If necessary, adjust the concentration of the cell suspension. Prepare the required number of slides and go to step 7.

5. For the cytocentrifugation of cells onto microscope slides, the cell density of the cell suspension is crucial. Therefore, after having discarded the supernatant, the pellet must be resuspended in the volume necessary to have approx 0.5 x 106 cells per milliliter (use only Hank's balanced salt solution [HBSS] or medium without serum; see Note 11). From this step, keep the cell suspension on ice.

6. Spot 100 ||L of cell suspension (i.e., 50,000 cells) per slide, at 800 rpm, 5 min (see Note 10). Check the cell density at the contrast phase microscope: cells must be evenly spaced, not overlapping each other, with well preserved cytoplasm. Adjust if necessary the concentration of the cell suspension (see Notes 10 and 12).

7. Fill the necessary number of Coplin jars (screwed cap) with -20°C absolute etha-nol and transfer the slides inside within 1 h from preparation (see Note 13). Slides can be kept at these conditions for several weeks.

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