A simple technique for quantifying apoptosis in 96-well plates.
BACKGROUND
Analyzing apoptosis has been an integral component of many biological studies. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell-damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and non-specific detection (i.e. "false positive"). To overcome the shortcomings of current methods that quantify apoptosis in vitro and to take advantage of the 96-well plate format, we present here a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification.


RESULTS
We compared our method and the conventional EB/AO method for quantifying apoptosis of suspension cells (Jurkat) and adherent cells (A375) under normal growth and apoptosis-inducing conditions. We found that our new EB/AO method achieved quantification results comparable to those produced using the conventional EB/AO method for both suspension and adherent cells.


CONCLUSIONS
By eliminating the detaching and washing steps, our method drastically reduces the time needed to perform the test, minimizes damage to adherent cells, and decreases the possibility of losing floating cells. Overall, our method is an improvement over the currently available techniques especially for adherent cells.
Ribble
Deborah
D
Dermatology Department, UCHSC at Fitzsimons, PO Box 6511, Aurora, CO 80045, USA. Deborah.Ribble@uchsc.edu
Goldstein
Nathaniel B
NB
Norris
David A
DA
Shellman
Yiqun G
YG
D000165:Acridine Orange / Q000494:pharmacology; D017304:Annexin A5 / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis; D001709:Biotechnology / Q000379:methods*; D002166:Camptothecin / Q000494:pharmacology; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000096:biosynthesis; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000494:pharmacology; D005189:False Positive Reactions; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D020287:In Situ Nick-End Labeling; D019169:Jurkat Cells; D009336:Necrosis; D013997:Time Factors
D017304:Annexin A5; D000972:Antineoplastic Agents, Phytogenic; D002843:Chromatin; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D004996:Ethidium; D000165:Acridine Orange; D002166:Camptothecin