Spontaneous apoptosis and ROS production in short term cultures. A) Peripheral blood mononuclear cells (PBMCs) from BLV-infected (n = 13) and control (n = 6) sheep were isolated and cultivated for 24 h. B cells were labeled using anti-IgM monoclonal antibody (clone 1H4) and FITC-conjugated rabbit anti-mouse and fixed in ethanol. After staining with PI, hypodiploid cells (sub-G1 population) considered to be apoptotic were quantified by flow cytometry. Data are presented as the means of apoptotic rates ± standard deviation. ** denotes the statistical significance according to the non-paired Student's t test p < 0.01. B) PBMCs from BLV-infected (n = 13) and non-infected sheep (n = 6) were seeded in 24-well plates at a density of 106 cells/ml and incubated for 30 min at 37°C with 10 μM of CM-H2DCFDA. After 24 h of culture, B cells were stained using anti-IgM monoclonal (clone Pig45) and Alexa Fluor 647-conjugated donkey anti-mouse antibodies. The intracellular ROS levels were determined by flow cytometry and are presented as the mean fluorescence intensities (± standard deviation) of cellular chloromethyldichlorofluorescein (CM-DCF) within B and non-B lymphocyte populations. *** denotes the statistical significance according to the non paired Student's t test p < 0.001. C) Correlation between apoptotic rates and ROS levels measured in ex vivo cultures. Apoptotic rates and ROS levels were determined by flow cytometry as described in panels A and B, respectively. A correlation coefficient R2 = 0.4097 was calculated from the linear regression analysis on percentages of apoptotic B cells and means of ROS fluorescence intensities. p < 0.05 denotes the statistical significance according to the non parametric Spearman test.