Comparison of apoptosis and mortality measurements in peripheral blood mononuclear cells (PBMCs) using multiple methods.
Death through apoptosis is the main process by which aged cells that have lost their function are eliminated. Apoptotic cells are usually detected microscopically by changes in their morphology. However, determination of early apoptotic events is important for in vitro (and ex vivo) studies. The main objective of the present study is to find the most sensitive method for apoptosis detection in human peripheral blood mononuclear cells (PBMCs) by comparing six different methods following five different means of immunological stimulation at 3 and 5 days. Each of six apoptosis quantification methods, except the trypan blue exclusion test, is a combination of two stains, one for the specific detection of apoptotic cells and the other for the unspecific detection of dead cells. Values for apoptosis and mortality were compared with a reference method. The choice of apoptosis detection method is more important following 3 days of stimulation than after 5 days of stimulation (P=2x10(-6) versus P=1x10(-2)). In contrast, we find mortality measurements following the different means of stimulation highly significant at both 3 and 5 days (F2.28=7.9, P=1.4x10(-6) at 3 days and F2.28=8.5, P=4.5x10(-7) at 5 days). Variation as a result of the combination of specific PBMC stimulation and the method used to detect apoptosis is reduced considerably with time (F1.58+3.7, P+3x10(-7) at 3 days to F=1.58=0.97, P=0.5 at 5 days). Based on Tukey's test, YO-PRO-1 is the most sensitive stain for apoptosis and, when combined with 7-AAD, provides an accurate measure of apoptosis and mortality. In conclusion, we propose YO-PRO-1/7-AAD as a new combination and low-cost alternative for the sensitive detection of early apoptosis.
Glisic-Milosavljevic
S
S
Max McGee National Research Center for Juvenile Diabetes and Human Molecular and Genetics Center, Medical College and Children's Hospital of Wisconsin, Milwaukee, WI 53226, USA.
Waukau
J
J
Jana
S
S
Jailwala
P
P
Rovensky
J
J
Ghosh
S
S
D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D049109:Cell Proliferation / Q000187:drug effects; D004396:Coloring Agents / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007958:Leukocyte Count / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000502:physiology*; D012016:Reference Values; D013053:Spectrophotometry / Q000379:methods; D013194:Staining and Labeling; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013936:Thymidine / Q000494:pharmacology
D000906:Antibodies; D018106:CD28 Antigens; D017252:CD3 Complex; D004396:Coloring Agents; D007376:Interleukin-2; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate; D013936:Thymidine