A new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells
✍ Scribed by H.J. Hasper; R.M. Weghorst; D.J. Richel; J.H. Meerwaldt; F.M.F.G. Olthuis; C.E.I. Schenkeveld
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 214 KB
- Volume
- 40
- Category
- Article
- ISSN
- 0196-4763
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✦ Synopsis
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 lympho-cyte 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 Ͻ 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.