Aquadead

PMN cell death was analyzed by treating human whole blood or isolated PMNs with different bacterial strains and LPSs. Human heparinized blood (100 or 500 μL) collected with lithium heparin was incubated for 2 hours at 37°C in agitation (200–300 rpm) with each treatment. Bacteria were tested at MOIs of 1, 10 or 100 bacteria/PMN. In the case of LPS or lipid A, blood samples were treated at concentrations from 3x10^-3 to 3x10¹ pmol/mL. After incubation, blood samples were lysed for 5–10 min in 900 μL of red blood cell lysis buffer (NH₄Cl 8.02 gm, NaHCO₃ 0.84gm and EDTA 0.37gm/L, pH 7.2). Cells were washed with ice cold PBS and re-suspended in 100 μL of Annexin V Binding Buffer (BD). 5 μL of Annexin V (BD) and 2 μL of AquaDead (Invitrogen) (diluted 1/20 in PBS) were added and incubated for 30 min on ice in the dark. Cells were washed once with ice cold PBS, re-suspended in 200 μL of paraformaldehyde 3% and incubated for 30 min at room temperature. Samples were then diluted 1:2 with PBS and acquired for analysis within 1 hour.

At the end of the tazemetostat treatment, we collected fresh tumors and peripheral blood mononuclear cells (PBMC) to assess the efficacy of the drug on EZH2 activity. PBMCs were exposed to RBC lysis buffer (NH₄Cl: 155 mM; NaHCO₃: 10 mM; EDTA: 0.1 mM) for 5 min on ice and washed with MACS buffer twice to remove red blood cells. Subsequently, PBMC were labeled with Aqua dead (L34966A; Invitrogen) for 30 min at 4 °C in PBS and subsequently fixed with fixation/permeabilization buffer (Ref: 00-5223-56, Invitrogen) during 30 min at 4 °C. Afterward, cells were intracellularly labeled with histone H3 antibody (ref: 12230S; Cell Signaling) and the specific H3K27me3 antibody (reference: 5499S) for 30 min at 4 °C in permeabilization buffer (Ref: 00-8333-56, Invitrogen). Finally, cells were washed and resuspended in MACs buffer for FACS analysis in BD LSR Fortessa. Gates were defined on single and live cells and the signal of H3K27me3 staining was calculated as a ratio of total H3 staining. In parallel, freshly resected tumors were fixed in formol/ethanol, and then embedded in paraffin; H3K27me3 staining was assessed using the tri-methyl-histone H3 (Lys27) (C36B11) with the previously described procedure. Brain delivery was not evaluated considering the extradural bone origin of chordomas.