Cytofix cytoperm

Enzyme immunoassays were used to measure cytokines in culture supernatants. 96 well plates were coated for 2 h at 37°C with anti-IFN-γ, anti-IL-17 or anti-GM-CSF in carbonate buffer 0.05 M pH 9.6. Culture supernatants or standards were incubated 2 h at 37°C. The plates were then incubated for 1h30 min with a secondary biotinylated antibody specific for each cytokine, followed by 20 min incubation with streptavidin-phosphatase alkaline at 37°C. Finally, plates were revealed by phosphatase alkaline substrate and absorbance was measured at 450/540 nm. For intracellular cytokine staining, cells were stimulated with different concentrations of MOG_35-55 (0, 10 and 100 μg) and treated with monensin (Golgiplug 1 μg/ml, BD Biosciences) at 37°C, in a humidified 5% CO2 atmosphere for 4 h. After staining of surface markers (TCR, CD4 and CD44), cells were fixed and permeabilized with Cytofix/Cytoperm and Perm/Wash buffer (e-Biosciences) according to the manufacturer’s instructions. Cells were then incubated with antibodies recognizing cytokines (IL-17, IFN-γ, GM-CSF) or isotype controls for 20 min and washed twice with Perm/Wash buffer before analysis. The production of IL-4, TNFα, IL-10 and IFN-γ by naive CD4 T cells was assayed using Cytometric Bead Array cytokine kit (BD Biosciences).

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).

Briefly, mouse spleen and draining lymph node (dLN) and kidney grinded to cell suspension resuspended in a red blood cell lysing buffer at 37°C for 2 min, followed by passing through a 70 mm cell strainer and centrifuged.
For intracellular IL-17A staining, cells were treated with 5 ng/ml PMA (Invitrogen) and 1 ng/ml ionomycin (Enzo Life Sciences, Inc.) for 5 h and then added at 10 ng/ml brefeldin A (Enzo Life Sciences, Inc.) 30 min before staining with CD4- FITC (eBiosciences, San Diego, United States). Next cells were stained with IL-17A-PE (eBiosciences) followed by permeabilization of the cells with Cytofix/Cytoperm (BD Biosciences, San Diego, United States).
For intracellular Foxp3-PE staining (Pan et al., 2014 (link)), cells were permeabilized with Cytofix/Cytoperm, and stained with Foxp3-PE (eBiosciences), followed by stain with CD4-FITC and CD25-APC (eBiosciences). Cells were detected by FACS Calibur Flow Cytometer (Becton Dickinson, San Diego, United States) and analyzed with the Flow Jo software.

For surface-molecule staining, cells were washed with staining buffer [PBS (Sigma-Aldrich, Darmstadt, Germany), 0.1% FCS, 200 mM EDTA] and subsequently stained with fluorochrome-conjugated monoclonal antibodies. After washing once with staining buffer, cells were analyzed with a Navios flow cytometer (Beckman Coulter, Brea, CA, USA). For intracellular staining, cells were washed with Perm/Wash™ buffer (eBiosciences, Waltham, MA, USA), fixed for 20 min at 4°C with Cytofix/Cytoperm™ (eBiosciences, Waltham, MA, USA) and stained in Perm/Wash™ buffer for 30 min at 4°C. After repeated washing with Perm/Wash™ buffer and staining buffer, cells were analyzed. A full list of monoclonal antibody combinations for identification of cell populations is provided in Table S2 in Supplementary Material. Ensuing evaluation of flow cytometric data was performed with Kaluza software 1.5a (Beckman Coulter, Brea, CA, USA).