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Simultaneous determination of apoptosis and surface antigen expression in tumor adherent cells
F Barzanti1, W Zoli, M D Susino
1Department of Medical Oncology, Morgagni-Pierantoni Hospital, Forli, Italy.
Abstract:
Apoptosis is a physiological, gene-directed form of cell death aimed at controlling cell proliferation in several biological conditions. It plays a crucial role in modulating tissue growth during embryonic development, cell turnover in adult life, and it seems to be the most frequent mechanism of tumor cell deletion by chemotherapy. Flow cytometry is a widely-used technique for checking apoptosis, permitting a multiparametric analysis. It is possible to follow the alterations occurring in the nucleus, mitochondria and plasmatic membrane during the different apoptotic stages using probes such as LDS-751, JC-1 or Annexin V. The potential of these probes to identify the early or late stages of apoptosis has been widely investigated in cells growing in suspension. In order to assess apoptosis in adherent cells, we tested a combination of fluorescein diacetate (FDA), a substrate for non specific esterase whose activity decreases during the early phase of apoptosis, and trypan blue in MCF-7 human breast cancer cells. Apoptotic cells showed a decrease in the green fluorescence emitted by fluorescein, the product of FDA hydrolysis, whereas necrotic cells emitted a red fluorescence due to the trypan blue staining. FDA-trypan blue double-staining was used to investigate the different kinetics of apoptosis induced by taxol, camptothecin and UV-B irradiation in MCF-7 cells. This method is rapid and simple, and can be used for monitoring the process of apoptosis from early stages in adherent cells, for the physical separation of apoptotic and live cells, and for immunophenotyping, including Fas expression.
Insights
This study introduces a simple fluorescein diacetate (FDA) and trypan blue double-staining method to detect early apoptosis in adherent cancer cells. This technique aids in monitoring apoptosis kinetics and cell separation.
Area of Science:
- Cell Biology
- Biochemistry
- Cancer Research
Background:
- Apoptosis is a crucial physiological process for controlling cell proliferation and tissue homeostasis.
- Chemotherapy often eliminates tumor cells via apoptosis.
- Assessing apoptosis in adherent cells presents challenges compared to suspension cells.
Purpose of the Study:
- To develop and validate a novel method for detecting early apoptosis in adherent cancer cells.
- To investigate the kinetics of apoptosis induced by various agents in breast cancer cells.
- To enable physical separation and immunophenotyping of apoptotic and live adherent cells.
Main Methods:
- Utilized fluorescein diacetate (FDA) and trypan blue double-staining on MCF-7 human breast cancer cells.
- Monitored changes in green fluorescence (from FDA hydrolysis) and red fluorescence (from trypan blue uptake).
- Applied the method to study apoptosis induced by taxol, camptothecin, and UV-B irradiation.
Main Results:
- Apoptotic cells exhibited decreased green fluorescence, while necrotic cells showed red fluorescence.
- The FDA-trypan blue method effectively distinguished between live, apoptotic, and necrotic MCF-7 cells.
- Demonstrated differential apoptosis kinetics induced by taxol, camptothecin, and UV-B irradiation.
Conclusions:
- FDA-trypan blue double-staining is a rapid and simple method for monitoring early apoptosis in adherent cells.
- This technique facilitates the physical separation of apoptotic and live cells for further analysis.
- The method is suitable for immunophenotyping, such as Fas expression analysis, in apoptotic cells.