FAN

To develop a method permitting fixation and permeabilization of samples after staining to measure apoptosis and necrosis by flow cytometry
Conclusion:
The described method resulted versatile, simple to use and suitable for a variety of in vitro or ex vivo assays aimed at detecting phenotypic characteristics of apoptotic and necrotic cells, including the analysis of intracellular markers that requires permeabilization of plasma membranes. Cells treated with the method can be fixed for a safe flow cytometry analysis of infected samples or for the comparative analysis of multiple samples in high-throughput settings. This is a potentially useful application of the method, since high-throughput settings could not be planned before for the multi-parametric analysis of apoptosis by flow cytometry due to limited time frame required to analyze sample treated with A-V/Pi. Lastly, the possibility to perform multi-parametric analyses including intracellular markers of dying cells offers the opportunity for in-depth analyses of mechanisms of apoptosis or pyroptosis that may lead to necrosis or pyronecrosis in a wide field of applications. 1. Instrument calibration, 2. Use of different instruments (FacsCalibur, Gallios), 3. FSC and SSC were controlled using human PBMC, T cell clones and macrophage lines, 4. Usage of isotype specific MoAb conjugated with the same fluorochromes of Ag specific MoAb 5. Compensation performed using single stained samples that were also used to confirm automatic compensation, when applied. 3. Numerous experimental repetitions, 4. Antibodies verified with existing methods prior to considering them for use, 5. Comparison of results (when possible) with preexisting methods