Transcription factor fixation permeabilization concentrate and diluent solutions

Immunostaining was performed as previously described on single cell suspensions derived from murine aortas to characterize immune cells [15 ]. To block Fc receptors, an unconjugated anti-CD16/32 antibody (clone 2.4G2, BD Bioscience) was used for mouse samples. Living cells were selected using Fixable Viability Dye-eFluor™ 780 (1:2000, eBioscience) and different cell populations were defined using anti-mouse fluorochrome-conjugated antibodies (Supplementary Table S1). Antibody staining of transcription factors and cytokines was performed using transcription factor fixation/permeabilization concentrate and diluent solutions and cytofix/permeabilization solutions, respectively (BD Biosciences). Flow cytometry analysis was performed on a Cytoflex S (Beckman Coulter) and the acquired data were analyzed using FlowJo software (version 10.7).

In vitro polarized macrophages, treated with 3GA-494 or 3GA-ctrl, were analyzed with flow cytometry to determine macrophage polarization phenotypes. Fc receptors were blocked using TruStain FcX (Biolegend) and an unconjugated anti-CD16/32 antibody (clone 2.4G2, BD Bioscience) for human and murine samples, respectively. Living cells were selected using Fixable Viability Dye eFluor 780 (1:2,000, eBioscience), and different cell populations were defined using anti-human and anti-mouse fluorochrome-conjugated antibodies. In human macrophages, the number of proinflammatory macrophages (CCR7⁺CD86^hi) or anti-inflammatory macrophages (CD206⁺) was quantified and shown as a percentage of positive cells within live CD45⁺CD11b⁺ cells. For murine macrophages, intracellular iNOS and Arg1 were stained using transcription factor fixation/permeabilization concentrate and diluent solutions (BD Biosciences). The number of proinflammatory macrophages (iNOS⁺) or anti-inflammatory macrophages (Arg1⁺) was quantified and shown as a percentage of positive cells within live CD11b⁺F4/80⁺ cells. MFI per cell was also quantified. Fluorescence-activated cell sorting analysis was performed on a Cytoflex S (Beckman Coulter), and the acquired data were analyzed using FlowJo software.