2.3 Mitochondrial function, cell death and morphology by microscopy
2.3.1 Fluorescence assessment for Mitochondrial activity .
Staining against Tetramethylrhodamine methyl-ester (TMRM - was used to assess mitochondrial membrane potential, ΔΨm) and Hoechst were assessed as a reliable marker of mitochondrial function and chromatin condensation respectively. Buffy coat was first rinsed in PBS (pH=7.4) for 5 min and then incubated with TMRM (1:200) in PBS for 30 min at 37oC. Thereafter, the mixture was washed and incubated with Hoechst (1:500) in PBS for 5 min at room temperature, and rinsed in PBS (pH 7.4) for 5 min per step. The mixture was then mounted with Vectashield and the image acquisition and processing were made with a microscope (Iroscope) fitted with a digital camera and imaging system using top view acquisition software. Fluorescent photomicrographs were obtained and the number of reactive cells to TMRM and Hoechst per field was determined in ten fields per sample, the results are shown by measurement of the densitometry (relative units) or the number of cells.
2.3.2 Fluorescence assessment for Apoptosis . Staining against annexin-V and propidium iodide were assessed as a reliable marker of apoptosis and necrosis respectively. Buffy coat was first rinsed in annexin-buffer for 5 min and then incubated with annexin-V/FITC (1:200) in annexin-buffer for 20 min at RT. Thereafter, the mixture was washed and incubated with propidium iodide (1:400) for 5 min at RT, and rinsed in PBS (pH 7.4) for 5 min per step. The mixture was then mounted with Vectashield and the image acquisition and processing were made with a microscope (Iroscope) fitted with a digital camera and imaging system using top view acquisition software. Fluorescent photomicrographs were obtained and the number of reactive cells to annexin-V, PI or annexin-V/PI per field was determined in ten fields per sample, the results are shown by counting the number of positive cells.