Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D.
This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.
Schmid
Ingrid
I
Department of Hematology/Oncology, Los Angeles, California 90095, USA. schmid@mednet.ucla.edu
Uittenbogaart
Christel
C
Jamieson
Beth D
BD
D017209:Apoptosis / Q000502:physiology*; D001562:Benzimidazoles / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003609:Dactinomycin / Q000031:analogs & derivatives*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D007834:Lasers
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D; C017807:bisbenzimide ethoxide trihydrochloride