Live dead fixable aqua dead cell stain

Rhesus macaque peripheral blood mononuclear cells (PBMC) were isolated from EDTA blood as described previously [72 (link)]. These cells were stained with fluorochrome-labeled MHC-I tetramers obtained from either the NIH Tetramer Core Facility or MBL International Inc. according to a recently published protocol [84 ]. Up to 800,000 PBMC were incubated with titrated amounts of each tetramer at room temperature (RT) for 45 min and then stained with fluorochrome-labeled monoclonal antibodies (mAbs) directed against the surface molecules CD3 (clone SP34-2), CD8α (clone RPA-T8), CD28 (clone 28.2), CCR7 (clone 150503), CD14 (clone M5E2), CD16 (clone 3G8), and CD20 (clone 2H7). Amine-reactive dye (ARD; Live/DEAD Fixable Aqua Dead Cell Stain; Life Technologies) was also added to this mAb cocktail. After a 25-min incubation at RT, the cells were washed with Wash Buffer (Dulbecco’s PBS with 0.1% bovine serum albumin and 0.45 g/L NaN₃) and then fixed with PBS containing 2% of paraformaldehyde. The configuration of the Special Order Product BD LSR II cytometer used to acquire the samples and the gating strategy employed to analyze the data have been detailed elsewhere [77 (link)]. In sum, we used FlowJo 9.6 to determine the percentages of live CD14⁻CD16⁻CD20⁻CD3⁺CD8⁺tetramer⁺ lymphocytes shown in Fig 2 and to delineate memory subsets within tetramer⁺ populations (Fig 4).

In order to analyse DSG1 and DSG3 specific B cells, B cells were isolated using Dynabeads Untouched Human B-cells kit (Life Technologies) according to the manufacturer's instruction. Then, purified B cells were incubated for 30 min at 4°C with histidine-tagged recombinant DSG1 or DSG3 (30 ng/μl). After washing, B cells were stained with anti-human IgG antibodies (BD Biosciences). Cells were then incubated with Fc Block (eBioscience), LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies), anti-human antibodies directed against CD19, CD27, IgM (BD Biosciences). Anti-histidine coupled with phycoerythrin (R&D Systems) was used to identify desmoglein-specific B cells.

Extracellular staining was performed in PBS 1× supplemented with 2 mM EDTA (Sigma-Aldrich) and 1% FCS. All extracellular staining was performed for at least 30 min at 4 °C. Dead cell staining (LIVE/DEAD Fixable Aqua Dead Cell Stain or Blue Dead Cell Stain, Life Technologies) was performed in PBS 1× according to the manufacturer’s recommendations. Samples were acquired with a FACS Fortessa X20 (BD Biosciences) or sorted with FACS ARIA III (BD Biosciences) using BD Diva v.9.0. All data were analyzed with FlowJo v.10.8.1 software. For the unsupervised analysis, t-SNE plots were generated with the dedicated plugin in FlowJo software. Dimensional reduction was performed on singlets/nonautofluorescent/live dead⁻/lineage⁻/CD19⁻/CD11c⁺ and/or SiglecH⁺ cells. Dimension reduction was calculated with the following markers: B220, BST2, CD11b, CD11c, CCR9, CX3CR1, Ly6D, SiglecH, tdT, XCR1 and Zbtb46-GFP. We used the HyperFinder plugin of FlowJo for the generation of unsupervised gating strategy. The calculation was performed on singlets/nonautofluorescent/live dead⁻ cells. We defined the pDC population for the calculation as being lineage⁻/CD19⁻/CD11b⁻/XCR1⁻/CD11c^low/BST2^high/tdT⁺. The software calculated the best and fastest gating strategy with the following markers: B220, BST2, CD11b, CD11c, CD19, CCR9, CX3CR1, lineage (CD3/Ly6G/NK1.1), Ly6D, SiglecH and XCR1.