Apoptosis assessment by the DNA diffusion assay.
A simple, sensitive, and reliable DNA diffusion assay for quantification of apoptosis is based on the principle that nuclear DNA of apoptotic cells have abundant alkali-labile sites and under alkaline conditions small pieces of DNA thus generated diffuse in agarose, giving the appearance of a halo if stained with a sensitive fluorescent dye such as YOYO-1. The protocol for detection of apoptosis described here is tested for estimation of apoptosis in human leukocytes (mostly neutrophils) incubated at 37 degrees C for 24 h. Cells were mixed with agarose, microgels were made, and cells were lysed in a solution of high salt, detergents, and alkali. DNA was precipitated in microgels by ethanol and spermine. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a circular gradient of granular DNA with a dense central zone and a lighter and hazy outer zone, giving the overall appearance of a halo.
Singh
Narendra P
NP
Department of Bioengineering, University of Washington, Seattle, USA.
D000818:Animals; D017209:Apoptosis; D001583:Benzoxazoles / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D003584:Cytological Techniques; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007962:Leukocytes / Q000473:pathology*; D008297:Male; D009336:Necrosis; D011806:Quinolinium Compounds / Q000494:pharmacology; D051381:Rats; D012685:Sepharose / Q000737:chemistry; D013094:Spermatozoa / Q000378:metabolism; D013696:Temperature; D013950:Thymus Gland / Q000378:metabolism; D013997:Time Factors
D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA; D012685:Sepharose