Live deadfixable dead cell stain kit far red

Whole
blood stimulations were performed as described above, except that
cells were stimulated for 2 h 37 °C, 5% CO₂, followed
by 4 h stimulation in the presence of Brefeldin A (BD Bioscience)
and Monensin (BD Bioscience) according to manufacturer’s recommendations
to block cytokine secretion. Subsequently, red blood cell lysis is
performed as described above and cells are stained with LIVE/DEAD
Fixable Dead Cell Stain Kit-Far Red (Invitrogen) according to manufacturer’s
recommendations. To allow intracellular cytokine staining, the cells
are treated with Cytofix/Cytoperm kit (BD Bioscience) according to
manufacturer’s recommendations. The following markers were
used to identify cell subsets: CD45-BV510 (BD Bioscience), CD3-APC-Cy7
(BD Bioscience), CD14-PeCy7 (Biolegend), and CD66b-BV421 (BD Bioscience).
Each sample divided was separately analyzed for the presence of IL-6
(IL-6-PECF594, Clone: MQ2-13A5, BD Bioscience), IL-8 (IL-8-PECF594,
clone: G265–8, BD Bioscience), or TNF-α (TNF-α-PECF594,
clone: MAB11, BD Bioscience). Flow cytometry data were collected on
a BD LSR II and analyzed using FlowJo 10.2 Software (Figure S2). Dead cells and CD45 negative events were excluded
from the data analysis.

Isolated
leukocytes (5 × 10⁵, 100 μL) were plated per
well of a sterile 96-well round-bottom plate (Falcon, Corning) and
mixed with 5 μL of BODIPY-labeled polymersomes in a cell:particle
ratio of 1:5 or 1:50. A polymer amount of 0.385 mg was estimated to
correspond to 5 × 10⁹ polymersomes. To block particle
uptake, leukocytes were incubated with Cytochalasin D (Sigma) at a
final concentration of 10 mM for 15 min at 37 °C, prior to adding
polymersomes. The cells were incubated with polymersomes for 30 or
60 min at 37 °C, 250 rpm. Subsequently, the cells were stained
with LIVE/DEAD Fixable Dead Cell Stain Kit-Far Red (Invitrogen) according
to manufacturer’s recommendations and followed by fluorescent
labeling of cell markers: CD45-BV510 (clone HI30, BD Biosciences),
CD14-PE-Cy7 (clone HCD14, Biolegend), CD66b-BV421 (clone G10F5, BD
Biosciences), CD56-PE (clone C5.9, Cytognos, Spain), and CD3-APC-Cy7
(clone SK7, BD Biosciences). Flow cytometry data were collected on
a BD LSR II (BD Biosciences) and analyzed using FlowJo 10.2 Software
(Figure S1). Dead cells and CD45 negative
events were excluded from the data analysis.