Live dead fixable far red dead cell stain kit

All general reagents and chemicals were purchased from Sigma–Aldrich, unless otherwise specified. Supplements and media for cell culture were from Invitrogen. Antibodies were purchased from Cell Signaling Technology (anti-Sirt6, catalog no.: 12486; anti-p27, catalog no.: 3688; anti–histone H3, catalog no.: 4620; and antiubiquitin, catalog no.: 3936), Abcam (anti-TNFα, catalog no.: ab183218 and anti-Sirt6, catalog no.: ab191385), or Sigma–Aldrich (anti–beta actin, catalog no.: A5441 and anti–alpha tubulin, catalog no.: T6074). Antibodies for flow cytometry were purchased from Thermo Fisher Scientific (LIVE/DEAD Fixable Far Red Dead Cell Stain Kit; catalog no.: L10120), BioLegend (CD19-APCCy7, catalog no.: 115530; T-cell receptor beta (TCRβ)-APCCy7, catalog no.: 109220; Ly6G-APCCy7, catalog no.: 127624; CD11b-BV510, catalog no.: 101245; and ZOMBIE-AQUA, catalog no.: 423102), or Millipore (F4/80-PE, catalog no.: MABF1530).

PBMCs and isolated monocytes were stained with LIVE/DEAD Fixable Far-Red Dead Cell Stain Kit (Thermo Fisher Scientific), Brilliant Violet 421 anti-human CD45 (2D1), PE anti-human CD63 (H5C6), Alexa Fluor 647 anti-human CD14 (63D3), PE/Cyanine5 anti-human HLA-DR (L243), and PE/Cyanine7 anti-human CD162 (KPL-1) or their respective mouse IgG isotype control antibodies (all from BioLegend). ECs were stained with LIVE/DEAD Fixable Far-Red Dead Cell Stain Kit, and Brilliant Violet 605 anti-human CD31 (WM59) or the respective mouse IgG1 isotype control antibodies (BioLegend). Heat treated monocytes or ECs were prepared by exposure to 60 °C for 10 min and used to separate live cells and dead cells on the histograms. Staining was performed in flow cytometry buffer (PBS supplemented with 2% P-FBS and 0.5 mM EDTA) for 30 min at ambient temperature. Unstained cells and cells stained with single antibodies were prepared to set a suitable compensation matrix to correct for fluorescent spillover. After 2× washes with flow cytometry buffer, cells were analyzed using CytoFLEX Flow Cytometry instrument (Beckman Coulter). Data analysis was performed using FlowJo, version 10.