Brilliant violet stain buffer

For ex vivo cytokine analysis, cells were first treated with a 1× cell activation cocktail of PMA/ionomycin/Brefeldin A (Biolegend for 5 h in complete RPMI media. 1 × 10⁶ cells from single cell suspensions were stained with anti-CD16/CD32 Fc block (1:200, BD Biosciences) and fixable viability 700 dye (1:10,000, BD Biosciences) in 1× PBS for 15 min. at 37 C prior to surface and intracellular staining with primary antibodies. Surface staining commenced in 200 μl FACS buffer supplemented with a 1:4 dilution of Brilliant Violet stain buffer (BD Biosciences) and fluorescently conjugated antibodies from the table below for 30 min. at 4 C in the dark; all are mouse reactive antibodies unless otherwise indicated. Following surface staining cells were washed and fixed in either 2% PFA or Fix/Perm buffer (eBioscience) for 20 min. at 4 °C for intracellular stain. Fix/Perm buffer was washed with 1× perm wash buffer (eBioscience) and intracellular proteins were stained with fluorescently conjugated ICS antibodies in perm wash buffer for 30 min at 4 °C in the dark. Cells were washed and resuspended in 1× PBS prior to acquisition on a BD Fortessa or LSRII flow cytometer (BD Biosciences). Please refer to Supplementary Table 1 for a complete list of antibodies used.

Human PBMCs (healthy and SLE) were isolated using BD Vacutainer®, CPT^™ mononuclear cell preparation tubes (BD Biosciences, NJ, USA) by density gradient centrifugation at 1700 g, deceleration = 6, for 25 min at 25˚ C. PBMCs were obtained, washed two times and resuspended as 1x10⁶ cells per 100 μL in Brilliant Violet stain buffer (BD Biosciences, NJ, USA). Cells were Fc receptor blocked for 15 minutes (TrueStain FcX, Biolegend) on ice and stained with various antibody panels. Cells were then washed and fixed by resuspending in 1% PFA. Data was collected using a BD FACS LSRII and analyzed using FlowJo version 10 (FlowJo LLC, OR, USA). Cells were stained with anti- CD3/CD19/CD20/CD56/CD45 (Lin^-) antibodies and further stained for CD7^-/HLADR⁺/CD141⁺/CD11c⁺/CD14⁺/CD16⁺ to specifically define gates for monocytes and dendritic cells (mDC1 and mDC2). LDGs were identified as Lin^-/ HLADR^-/CD15⁺/CD11b⁺/CD33⁺/CD14^-/CD15⁺ and monocytic MDSCs stained as Lin^-/ HLADR^-/CD15⁺/CD11b⁺/CD33⁺/CD14⁺/CD15^-. All cell types were stained for LOX-1.

Single-cell suspensions isolated from rhesus macaque, human, or mouse tissues were prepared for flow cytometry by first incubating the cells with the appropriate Fc block (rhesus/human: Human TruStain FcX, Biolegend or mouse: purified rat anti-mouse CD16/CD32, BD Biosciences) in combination with a viability dye (Live/Dead Aqua or Violet, ThermoFisher Scientific). Following the Fc block and viability staining step, panels of fluorescently conjugated antibodies were used to stain markers of interest. Details on markers targeted, antibody clones used, and fluorescent conjugates used can be found in Supplementary Data 2. All cells were stained in a 1:1 mix of FACS buffer (1% FBS 2 mM EDTA in PBS) and Brilliant Violet Stain Buffer (BD Biosciences). Staining for human and rhesus samples was conducted at room temperature. Staining for mouse samples was conducted at 4 °C.

Cryopreserved PBMC specimens were thawed and washed, and counts and viability were assessed using the Countless Automated Cell Counter system (Invitrogen, Carlsbad, CA). Cells were washed and stained in Brilliant Violet Stain Buffer (BD Biosciences, San Jose, CA) at room temperature for 15 min in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry data acquisition. Intracellular staining for IFNγ and promyelocytic leukaemia zinc finger (PLZF) were performed in Perm/Wash (BD Biosciences) at room temperature for 15 min in the dark. mAbs used in flow cytometry: CD3 AF700, CD3 PerCP-Cy5.5 (both clone UCHT1), CD4 BV605 (clone RPA-T4), CD8 BV711 (clone RPA-T8), CD38 APC-H7 (clone HB7), CD69 AF00 (clone FN50de), CD161 BV421 (clone DX12), CCR6 BV786 (clone 11A9), HLA-DR APC (clone L243), IFNγ APC (clone B27), and PD-1 PE-Cy7 (clone EH12.1) were all from BD Biosciences, TCR Vα7.2 PE, TCR Vα7.2 PerCP-Cy5.5 (clone 3C10) were from Biolegend (San Diego, CA, USA), and PLZF APC was from R&D Systems (Minneapolis, MN). Live/dead aqua fixable cell stain was from Life Technologies (Eugene, OR, USA) and added to the cells together with the cell surface antibodies. Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar, Ashland, OR, USA).