Cd40l b220

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)^dimEth^bright cells.

At euthanization, pancreas, spleen, kidneys with adrenals, liver and thymus were removed and cleaned of connective tissue before weighing. Histopathology is included in the supplementary methods. For flow cytometry, the spleens were mashed in 3 mL PBS solution on ice. Leukocyte populations were characterized by flow cytometric analysis with different combinations of fluorochrome-labeled antibodies (diluted 1:80–1:100; BD PharMingen, San Diego, CA) including CD4-CD8-CD25 (PE-PerCP-FITC), CD40L-B220 (PE-FITC) and Mac3-Gr1 (PE-FITC) in females. For males, we used CD4-CD8-CD25 (PE-PerCP-FITC), CD40-LB220 (PE-FITC) and F4/80-Gr1 (PE-FITC). CD4-CD8-CD25-Mac3-CD45R (V450-APCH7-APCA-FITC-PE), CD40L-B220 (PE-FITC), CD5-CD24 (PE-FITC) and CD44-CD40 (PE-FITC) were used in antibiotic treated females. Isotype matched irrelevant antibodies were used as controls. Following addition of the antibodies, the cells were incubated at 4°C in the dark for 30 min. The cells were washed and enumeration performed on a Becton Dickinson LSRII Flow Cytometer (BD Biosciences, San Jose, CA) in which log fluorescence intensity was read and a forward scatter threshold high enough to eliminate red blood cells. Ten thousand cells were counted for each sample. Analysis was done using FlowJo software (FlowJo LLC).