Tcs sp5 germany

Apoptosis induced by P. zopfii in bMECs was interpreted by annexin V-FITC and PI staining method. The bMECs (1 × 10⁵ cells/mL) were cultured as mentioned above. These cells were infected with GTI and GTII for 4 h, 12 h and 24 h. After treatment, the coverslips were washed thrice with ice cold PBS and stained with FITC conjugated annexin-V mixture at room temperature (30 min), then bMECs were stained with PI (5 min) after twice washing with cold ice PBS, finally, stained bMECs were analyzed under confocal laser-scanning microscope (LEICA TCS SP5-Germany) with an excitation/emission 488/ 525 nm for FITC and excitation/emission 488/620 nm for PI. The proportion of the positive apoptotic bMECs were expressed as percentage of counted cells.

The intracellular reactive oxygen species (ROS) in bMECs was evaluated using probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA). The bMECs (1 × 10⁵ cells/mL) were treated with GTI and GTII (5 × 10⁵ cells/mL) for 4 h, 12 h and 24 h. Infected cells were washed with PBS and treated with DCFH-DA (5 μmol/L) (20 min). After discarding the supernatant the treated cells were resuspended in PBS (two times). Fluorescence assays were measured and quantified by confocal laser-scanning microscope (LEICA TCS SP5-Germany) using excitation/emission 525/610 nm and also analyzed by flow cytometry (Beckman, Fullerton, California, USA) at excitation/emission 488/525 nm.