The spleens were collected upon euthanasia, and the flow cytometric analysis was performed following our previous publication [29 ]. Briefly, the spleens were mashed in 3 mL PBS solution on ice. Leukocyte population in NOD mice were characterized with different combinations of fluorochrome-labeled antibodies (diluted 1:80-1:100; BD PharMingen, San Diego, CA) including cluster of differentiation (CD) 4-CD8-CD25 (PE-PerCP-FITC), CD40L-B220 (PE-FITC) CD40-CD44 (PE-FITC) and CD5-CD24 (PE-FITC).
For the B6C3F1 females, the antibodies included IgM-CD3 (FITC-PE), CD4-CD8-CD25 (PE-PerCP-FITC), NK1.1-CD3 (PE-FITC), and Gr-1-Mac-3 (FITC-PE). In addition, the analyses of mitochondrial transmembrane potential (ΔΨm) and reactive oxygen species (ROS) generation were performed following our previous publication [18 (link)]. Thymocytes (1 × 106 cells/ml) were stained for 15 min with 40 nM 3,3’-dihexyloxacarbocyanine (DiOC6(3); Life Technologies) and 2 μM hydroethidine (HE, Life Technologies) for assessing ΔΨm and ROS generation. Following excitation at 488 nm (250 mW), emission was monitored through a 530/30 nm bandpass filter for DiOC6(3) and 575/26 nm bandpass filter for HE, and then logarithmic amplification was used to detect the fluorescence. The late apoptosis cell population was represented by DiOC6(3)dimEthbright cells.
For the B6C3F1 females, the antibodies included IgM-CD3 (FITC-PE), CD4-CD8-CD25 (PE-PerCP-FITC), NK1.1-CD3 (PE-FITC), and Gr-1-Mac-3 (FITC-PE). In addition, the analyses of mitochondrial transmembrane potential (ΔΨm) and reactive oxygen species (ROS) generation were performed following our previous publication [18 (link)]. Thymocytes (1 × 106 cells/ml) were stained for 15 min with 40 nM 3,3’-dihexyloxacarbocyanine (DiOC6(3); Life Technologies) and 2 μM hydroethidine (HE, Life Technologies) for assessing ΔΨm and ROS generation. Following excitation at 488 nm (250 mW), emission was monitored through a 530/30 nm bandpass filter for DiOC6(3) and 575/26 nm bandpass filter for HE, and then logarithmic amplification was used to detect the fluorescence. The late apoptosis cell population was represented by DiOC6(3)dimEthbright cells.