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Cell mediated cytotoxicity assessment by relative changes in viable target absolute counts
G N Topakas1, I N Karchilaki, N T Cacoullos
1Department of Immunology, G. Gennimatas General Hospital of Athens, Greece. svoronos@hotmail.com
Immunopharmacology and Immunotoxicology
|November 6, 2001
Summary
This study introduces a flow cytometry method to measure cell killing by NK and CTL cells. The technique accurately quantifies viable target cells, proving effective in assessing cytotoxic activity in various assays.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Assessing cell-mediated cytotoxicity is crucial for understanding immune responses and developing therapies.
- Existing methods for quantifying cytotoxic activity often rely on beads or standards, which can introduce variability.
- A need exists for a sensitive, accurate, and efficient method to measure absolute counts of viable target cells after effector cell exposure.
Purpose of the Study:
- To develop and validate a novel flow cytometry-based method for measuring absolute counts of viable target cells.
- To assess the cytotoxic activity of Natural Killer (NK) cells and Cytotoxic T Lymphocytes (CTLs) using this new methodology.
- To evaluate the method's performance in different immunological contexts, including allogeneic stem cell transplantation and autologous assays.
Main Methods:
- Utilized Calcein-AM staining to identify viable target cells and 7-Amino Actinomycin D (7-AAD) to exclude dead cells.
- Employed a Cytoron Absolute flow cytometry system to determine absolute cell counts (cells/microliter) without beads or standards.
- Correlated the percentage change in viable target cell counts with effector-to-target ratios using linear regression.
Main Results:
- The method demonstrated high precision, with a Coefficient of Variation (CV) below 6% for initial cell concentrations.
- Positive cytotoxicity (p < 0.05, r2 > 0.9) was observed in 15/20 allogeneic mixed lymphocyte cultures (MLC) and all NK assays.
- Negative or inconclusive results (p > 0.05, r2 < 0.4) were found in 5/20 allogeneic MLC and all autologous MLC assays.
- The method is sensitive enough to estimate CTL frequencies higher than 1:100.
Conclusions:
- The developed method combines absolute cell counting with flow cytometry for precise assessment of viable targets.
- It allows for the analysis of cell behavior during cytotoxic phenomena using small cell amounts and offers excellent sensitivity.
- This approach provides a valuable tool for evaluating cytotoxic activity in various immunological settings, including clinical applications like stem cell transplantation.