Novel single-platform multiparameter FCM analysis of apoptosis: Significant differences between wash and no-wash procedure.
FCM is a generally accepted tool to analyze apoptosis. Unfortunately, the cell preparation of all commercial kits available includes cell washing known to cause cell loss which is most likely to affect apoptotic cells in particular. To address this, we developed a seven-color single-platform no-wash analysis technique and compared the results with those from an analogous procedure including cell washing. A five-color mAb cocktail was employed to address target cells by surface labeling, Yo-PRO-1® and DAPI were used to discriminate apoptotic and necrotic from viable cells. Cells were quantified on the basis of internal-standard fluorescent beads. Jurkat cells ACC 282 treated with camptothecin were employed to establish the staining procedure, which was then applied to blood cells collected by extracorporeal apheresis and treated with UV irradiation. Data evaluation showed that although each method by itself was highly reproducible (R(2) = 0.973), the numbers of apoptotic cells detected with the no-wash procedure were significantly higher than those obtained after cell washing (P = 6.6 E(-5), Wilcoxon Test). In addition, the observed differences increased with higher cell numbers (Bland and Altmann). We conclude that the described test is a feasible and reliable tool for apoptosis measurement and it provides results that are definitely closer to the truth than those obtained from kits that require cell washing.
Stemberger
Julia
J
Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
Witt
Volker
V
Printz
Dieter
D
Geyeregger
René
R
Fritsch
Gerhard
G
D000911:Antibodies, Monoclonal / Q000737:chemistry; D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity; D017209:Apoptosis / Q000502:physiology*; D002166:Camptothecin / Q000633:toxicity; D002452:Cell Count / Q000379:methods; D002469:Cell Separation; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells / Q000737:chemistry / Q000187:drug effects / Q000473:pathology; D015458:Leukemia, T-Cell / Q000097:blood / Q000473:pathology* / Q000628:therapy; D009336:Necrosis; D017893:Photopheresis; D015203:Reproducibility of Results; D012996:Solutions
D000911:Antibodies, Monoclonal; D000972:Antineoplastic Agents, Phytogenic; D012996:Solutions; D002166:Camptothecin