The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single-cell level.
The present study describes the improvement of a technique, the alkaline-halo assay (AHA), for the assessment of DNA single-strand breakage at the single-cell level. AHA involves a series of sequential steps in which cells are embedded in melted agarose and spread onto microscope slides, incubated in a high-salt alkaline lysis solution, then in a hypotonic alkaline solution and, finally, stained with ethidium bromide (EB). Under these conditions, single-stranded DNA fragments diffuse radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nuclear remnants: the area of the halo is a direct function of the extent of DNA strand scission. These phenomena can be conveniently monitored with a fluorescence microscope and quantified by image-processing analysis. The behaviour of single-stranded DNA fragments under the conditions of the modified assay, called fast halo assay (FHA), is essentially the same as in AHA. The modifications consist in the simplification of the lysis, denaturation and staining procedures, and allow, as compared with AHA, the preparation of samples within 15 min, with a two-third reduction in total processing time, using only two reagents to promote DNA extraction and staining: NaOH and EB. A variation of the FHA operating at non-denaturing conditions to discriminate apoptotic cells from non-apoptotic cells bearing DNA single-strand breaks is also illustrated. To benchmark FHA sensitivity and reliability, the DNA single-strand breaks (SSBs) resulting either from exposure of cultured mammalian cells to different DNA-damaging agents or from secondary apoptotic DNA cleavage, have been quantified and results compared with the outcomes of reference techniques run in parallel, namely AHA, comet assay and Hoechst 33342 staining. The results indicate that FHA has the same reliability and sensitivity of the reference assays, but presents the additional advantages of being inexpensive, more rapid and strikingly simple.
Sestili
Piero
P
Istituto di Ricerca sull'Attività Motoria, Via Sasso 75/G, Università degli Studi di Urbino "Carlo Bo", 61029 Urbino, Italy. sestili@uniurb.it
Martinelli
Chiara
C
Stocchi
Vilberto
V
D000468:Alkalies / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles; D001681:Biological Assay / Q000379:methods; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D020552:Comet Assay / Q000592:standards; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000302:isolation & purification; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology; D004996:Ethidium / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009152:Mutagenicity Tests / Q000191:economics; D009153:Mutagens / Q000145:classification / Q000633:toxicity*; D009691:Nucleic Acid Denaturation; D015203:Reproducibility of Results; D012492:Salts / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013997:Time Factors; D020298:U937 Cells; D014471:Umbilical Veins / Q000166:cytology
D000468:Alkalies; D001562:Benzimidazoles; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D007038:Hypotonic Solutions; D009153:Mutagens; D012492:Salts; D004996:Ethidium; C017807:bisbenzimide ethoxide trihydrochloride