Camel Leukocyte Apoptosis and Necrosis

Cell necrosis was measured by the dye exclusion assay [26 (link)]. Briefly, camel leukocytes were labeled with propidium iodide (PI) at a final concentration of 2 µg/mL (Calbiochem, Germany) and PI-fluorescence was measured by flow cytometry (detected in FL-3). PI-positive dead cells with permeable cell membranes were distinguished from PI-negative live cells. For the measurement of cell apoptosis, separated leukocytes (100µL in RPMI-1640 cell culture medium) were incubated with JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) in a 96-well microtiter plate [27 (link),28 (link),29 (link)]. JC-1 solution (100µL of 2μmol/L final concentration) was added to the cells in each well for 15 min (5% CO2) at 37 °C. Finally, labeled cells were washed twice with PBS, suspended in 200μL PBS, and analyzed on the Acuri C6 flow cytometer (BD Biosciences). Apoptotic cells with JC-1 monomers (increased green fluorescence) were distinguished from normal non-apoptotic cells with orange JC-1 aggregations (increased orange fluorescence in FL-2). Hydrogen peroxide (H2O2; 180 uM)) has been used to establish a positive control of apoptosis.