A new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells.
BACKGROUND
Human peripheral blood lymphocytes kept in culture after isolation die by an apoptotic process. Detection of apoptosis with labeled Annexin V to demonstrate loss of plasma membrane asymmetry is sensitive, specific, and easy using flow cytometry. This is true in lymphoblastic cell lines when combining Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). However, measurement of apoptosis by flow cytometry in isolated human lymphocytes using Annexin V-FITC/PI is disturbed by the presence of a variable percentage of erythrocytes in the isolated lymphocyte population. To overcome this problem, we have developed and tested a new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells.


METHODS
Peripheral blood lymphocytes are isolated by density gradient centrifugation. Nucleus-containing cells are selected using CD45-phycoerythrin (PE). The lymphocyte subset of interest is selected using CD4, CD8, or CD19 energy-coupled dye (ECD) labeling. Apoptosis is detected using Annexin V-FITC with 7-amino-Actinomycin-D (7-AAD) to distinguish early apoptotic from late apoptotic lymphocytes.


RESULTS
We have developed a new technique to detect apoptosis in isolated human peripheral blood lymphocyte subsets with good reproducibility, coefficient of variation < 17%.


CONCLUSIONS
We now have a validated tool to study apoptosis in subsets of isolated human lymphocytes to increase our knowledge of pathogenesis and therapies in lymphoreticular malignancies.
Hasper
H J
HJ
Department of Internal Medicine, Medical Spectrum Twente, Enschede, The Netherlands.
Weghorst
R M
RM
Richel
D J
DJ
Meerwaldt
J H
JH
Olthuis
F M
FM
Schenkeveld
C E
CE
D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D002478:Cells, Cultured; D003116:Color; D004396:Coloring Agents; D003609:Dactinomycin / Q000031:analogs & derivatives; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D016131:Lymphocyte Subsets / Q000737:chemistry / Q000166:cytology*; D011419:Propidium; D015203:Reproducibility of Results
D017304:Annexin A5; D015704:CD4 Antigens; D016827:CD8 Antigens; D004396:Coloring Agents; D005456:Fluorescent Dyes; D003609:Dactinomycin; D011419:Propidium; C025942:7-aminoactinomycin D; D017493:Leukocyte Common Antigens; D016650:Fluorescein-5-isothiocyanate