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Updated: Aug 9, 2026
Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity
Published on: December 17, 2013
Flow cytometric quantification of apoptosis and proliferation in mixed lymphocyte culture
Luis Marín1, Alfredo Minguela, Alberto Torío
1Immunology Service, University Hospital Virgen de la Arrixaca, Murcia, Spain.
Insights
This study introduces a novel method for analyzing mixed lymphocyte cultures (MLC), improving the reliable quantification of allogeneic immune responses by assessing both cell proliferation and apoptosis simultaneously.
Area of Science:
- Immunology
- Cell Biology
Background:
- Mixed lymphocyte culture (MLC) is a standard in vitro method for studying allogeneic immune responses.
- Current MLC methods face challenges in accurately identifying and quantifying responder cells, including apoptotic or proliferative cells.
Purpose of the Study:
- To develop and validate a novel, non-radioactive method for simultaneous determination of apoptosis and proliferation in MLC.
- To enhance the reliability of assessing allogeneic immunoresponses in vitro.
Main Methods:
- Peripheral blood mononuclear cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE).
- Unidirectional cultures were stimulated with allogeneic irradiated cells.
- Apoptosis was assessed using 7-aminoactinomycin D, with propidium iodide-stained cells used as an internal reference standard.
Main Results:
- CFSE labeling did not interfere with proliferation or apoptosis assessments.
- The novel method's apoptosis data correlated well with traditional monoclonal antibody techniques.
- Proliferation measurements showed good correlation with both [(3)H]-thymidine incorporation and direct counting of viable proliferative cells.
Conclusions:
- The developed method enables simultaneous, non-radioactive measurement of apoptosis and proliferation in MLC.
- This assay allows for detailed cell phenotype analysis, offering new insights into graft tolerance mechanisms in solid organ transplantation.
Background:
The one-way mixed lymphocyte culture (MLC) is the classic culture used for studying the allogenic immunoresponse in vitro, but stimulator and responder cell identifications and quantification of apoptotic or proliferative responder cells are unreliable.
Methods:
Peripheral blood mononuclear cells were labeled with 5- (and 6-) carboxy fluorescein diacetate succinimidyl ester (CFSE) and stimulated with allogenic unlabeled irradiated cells in unidirectional cultures. Apoptosis was determined by the 7-aminoactinomycin D technique, and the absolute number of each cell population was calculated by adding a fixed number of cells stained with propidium iodide as the reference standard for each test.
Results:
CFSE labeling of cells under different cultures did not affect the results of proliferation or apoptosis. Data of apoptosis obtained with this method were comparable to those of the monoclonal antibody technique, and the proliferation level determined by [(3)H]-thymidine incorporation or counting the number of proliferative living cells, as proposed in this method, showed a good correlation.
Conclusions:
The method presented in this report allows the simultaneous determination of apoptosis and proliferation in MLCs and the analysis of cell phenotype, thereby avoiding the use of radioactivity. This assay opens new perspectives for a better understanding of the mechanisms implied in the establishment or break of tolerance to the graft in solid organ transplants.
