Instrument fc 500 flow cytometer

Quantitative measures of viable, early, and late apoptotic and necrotic cells in ovaries and testis were obtained with the Annexin V-FITC (fluorescein isothiocyanate) Apoptosis Detection Kit (Abcam, № ab14085). This method is used to detect the early stages of apoptosis when translocation of phosphatidylserine (PS) groups from the inner to the outer leaflet of the plasma membrane occurs. Green fluorescence origins form cells bounded to the FITC-labeled Annexin V, while the red fluorescence origins form propidium iodide (PI). Thus, the distinction between necrotic cells (Annexin V-FITC–/PI +) and apoptotic cells (early apoptotic cells: Annexin V-FITC + /PI–; late apoptotic cells: Annexin V-FITC + /PI +) was enabled. Labeling was performed in dark according to the manufacturer’s protocol. The above-described cell suspension was analyzed in the Beckman Coulter Instrument FC 500 flow cytometer with a 488 nm argon laser. Fluorescence level results were examined with the CXP Analysis software for cytometric data.

The dissected organs isolated from specimens from each experimental group were mechanically fragmented with scissors and suspended in 100 µL of PBS (pH 7.4). Then, the intestine and hepatopancreas cells were separated by gentle shaking in a homogenizer (Minilys; Bertin Technologies). The cell suspension was washed using centrifugation at 1500 rpm for five minutes and the precipitate was suspended in 100 µL of PBS buffer.
For the quantitative measurements of cellular populations undergoing oxidative stress were used the Muse Oxidative Stress Kit (Merck Millipore, No. MCH100111). The assay is based on dihydroethidium (DHE), which upon reaction with superoxide anions undergoes oxidation, resulting in red fluorescence. According to the manufacturer’s protocol, the results were expressed as the percentage of two populations of cells: ROS negative (live cells) and ROS positive (cells exhibiting ROS). The measurements were performed using the Beckman Coulter Instrument FC 500 flow cytometer with a 488 nm argon laser.