Beckman cytomics fc 500

The spleens were cut into pieces, washed with RPMI-1640 culture medium, and centrifuged for 5 min at 1500r/min. After filtering with 100-mesh nylon mesh, the supernatant was discarded, and the spleen cells of each group were suspended with RPMI-1640 culture medium, and then rat spleen lymphocytes were isolated and collected by using a rat peripheral blood lymphocyte separation medium kit.
Isolated rat spleen lymphocytes were washed twice with PBS containing 1% fetal calf serum (FCS) and then resuspended in it at a concentration of 1 x 10⁶ cells/ml. One hundred microliters of the solution of each group was transferred into a 5 mL culture tube; then 5 μl of PE Mouse anti-rat CD4 mAb and FITC Mouse anti-rat CD25 mAb was added. The cells were gently vortexed and incubated for 30 min at room temperature in the dark. Next, 400 μl of PBS containing 1% FCS was added to each tube. The prepared samples were analyzed by flow cytometry (Beckman Cytomics FC 500) within 1 hour.

The cells were incubated with 3% bovine serum albumin (Sigma, USA) for 30 min to block nonspecific antigens. Then, they were incubated with the following monoclonal antibodies (BD, USA) for 30 min: CD29-APC, CD34-APC, CD44-FITC, CD45-PE, CD73-PE, CD90-FITC, and HLA-DR-FITC. After incubation, the cells were washed with PBS and analyzed in a flow cytometry analyzer (Beckman Cytomics FC500, Beckman Coulter, USA).

We used the ROS-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate to determine the intracellular ROS levels (H2DCFDA, SIGMA-ALDRICH Corp., St. Louis, MO USA). Following intermittent hypoxia/reoxygenation (IHR) stimulation, cells were incubated in the dark for 30 min with 5 M H2DCFDA and immediately analyzed using a flow cytometer set to 488 and 535 nm excitation and emission wavelengths, respectively (Beckman Cytomics™ FC500, Beckman Coulter, Brea, CA, USA).