Live dead yellow stain

PBMCs isolated from peripheral blood of human T1D patients and age-matched controls were stained with the following antibodies: CD3, CD14, CD16, CD19, CD56, CD74, and HLA-DR (all eBioscience, San Diego, CA) and matching isotype controls. Non-viable cells were excluded by using the fixable Live/Dead Yellow stain (Invitrogen) and monocytes were further defined as previously described [25 (link)]. Data acquisition was performed on a Gallios^™ flow cytometer (Beckman Coulter, Analis, Suarlée, Belgium) and analyzed using FlowJo^™ software (Treestar, Ashland, OR). For intracellular staining, PBMCs were stimulated with LPS and brefeldin A (eBioscience) for 18 h and then stained with the same antibody cocktail as described above followed by the addition of Cytofix/Cytoperm (eBioscience) and anti-human TNFα (eBioscience).

Peritoneal macrophages were collected [27 (link)], and preconditioned with 20 μM MIF antagonist (ISO-1, Sigma) or vehicle control for 24h before overnight culture in the presence or absence with 10 ng/ml murine recombinant IFN-γ (Peprotech, Rocky Hill, NJ) and 1 μg/ml LPS (Sigma).
For phenotypic analysis macrophages were seeded in ultra-low attachment plates at 2 × 10⁵ cells per well before preconditioning with ISO-1 and stimulation with LPS/IFN-γ as described above. Thereafter, 2 × 10⁵ cells were labeled with the following conjugated Abs: F4/80, CD11b, I-A^d (clone 39-10-8 for NOD), I-A^b (clone AF6-120.1 for C57BL/6), CD86 and matching isotype controls (all eBioscience). Non-viable cells were excluded by using the fixable Live/Dead Yellow stain (Invitrogen). Data acquisition was performed on Gallios^™ flow cytometer (Beckman Coulter) and the FlowJo^™ (Treestar) software was used for data analysis.

PBMCs from healthy donors were isolated by Ficoll gradient centrifugation from whole blood purchased from the New York Blood Center. Isolated cells were cultured in complete RPMI media, consisting of RPMI-1640 supplemented with 5 mM HEPES, 2 mM Glutamine, 50 μg/mL penicillin/streptomycin, 5 mM nonessential amino acids, 5 mM sodium pyruvate, and 10% fetal bovine serum (Thermo Fisher). PBMCs were incubated for 24 h in 6-well plates with IU1 (200 μM), b-AP15 (1 μM), or Romidepsin (0.1 μM). After washing with FACS buffer (PBS, 2% FBS), PBMCs were stained with the following cocktail of anti-human antibodies: CD3-FITC (BioLegend, San Diego, CA), CD4-Pacific Blue (BioLegend), CD25-PE-Cy7 (BioLegend), and CD69-Alexa Fluor 700 (BioLegend). In addition, LIVE/DEAD Yellow stain (Invitrogen, Thermo Fisher Scientific, Waltham, MA) was used to exclude dead cells. Staining was performed for 30 min on ice in FACS buffer. After washing, the samples were analyzed on a multi-laser flow cytometer LSR-II (BD Bioscience, San Jose, CA). Fluorescence compensation was performed with anti-mouse IgG (k) beads (BD Biosciences) stained with each antibody in a separate tube. The total content of PBMCs acquired by BD LSRII was analyzed with FlowJo (TreeStar, Cupertino, CA).