Hanks balanced salt solution (hbss)

The extracellular nucleotides were detected using SytoxGreen staining [20 (link)] Briefly, isolated from bone marrow cells neutrophils were transferred to the SytoxGreen dissolved in HBSS (PanEco, Moscow, Russia) 1:1000. Neutrophils were incubated in a black 96-well plate (SPL Life Sciences, Pocheon, Korea) in a concentration of 2 × 10⁵ cells per well at 37 °C and 5% CO₂ in the presence of 5 µM of A23187 for 3 h. The samples were analyzed using GloMax Microplate reader Multisystem (Promega, Madison, WI, USA) in a fluorescence mode, filter 490 nm.
Neutrophils were also loaded to the coverglass-bottom slides (Ibidi, Gräfelfing, Germany), incubated 3 h in presence or without of 5 µM of A23187, then fixed with 4% paraformaldehyde (Applichem, Darmstadt, Germany). The samples were stained with anti-histone H3 (citrulline R2 + R8 + R17) antibody (Abcam, Boston, MA, USA, ab5103) and the secondary goat-anti-rabbit PE-conjugated (Sigma-Aldrich, St. Louis, MO, USA, P9537). The nuclei were stained with Hoechst (PanEco, Moscow, Russia), dilution 1:1000.

Single-cell suspension samples were acquired separately from each mouse. Blood vessels were washed out by perfusion with 0.02% EDTA-PBS solution introduced through the right ventricle. The lungs were removed and sliced into 1–2-mm³ pieces. These were incubated in supplemented RPMI-1640 containing 200 U/ml collagenase and 50 U/ml DNase-I (Sigma-Aldrich, St Louis, MO) at 37°C for 90 min. The suspensions were washed three times in HBSS (Paneco, Russia) containing 2% fetal calf serum (FBS, General Electric, Boston, MA, USA) and antibiotics (Sigma, St. Louis, MO, USA). Next, the cells (50 x 10⁶ per dish) were incubated in 10 ml of medium (RPMI-1640, 10% FCS, 10 mM HEPES, 2 mM l-glutamine and antibiotics) in 90-mm-diameter cell culture-treated Petri dishes for 1 h at 37°C to achieve cell adhesion. Three repeated rounds of vigorous washing with warm antibiotic-free HBSS containing 2% FCS were performed to collect nonadherent cells. Two additional rounds of washing under the same conditions were then performed to get rid of residual medium. Cells then were resuspended in PBS, calculated, and proceeded for FACS analysis and cell sorting.

A Si₃N₄ cantilever (C-MSCT, Bruker, Billerica, MA, United States) with f₀ 4–10 kHz and k – 0.010 N/m was used for attaching the NGs. For better attachment of the cells, the cantilever was coated with poly-L-lysine (20 μL, 0.01%), then washed three times with a sterile normal saline solution (10 μL), and incubated (37°C, 20 min) with cell suspension (10 μL). After that, the cantilever with NGs was washed three times with PBS. The concentration of NGs was selected in a series of preliminary experiments (Pleskova et al., 2020 (link)). The prepared cantilever with cells was put on a control head.
The control signal of clear cantilever deflection was recorded as described in paragraph 2.1.5. A suspension of S. aureus 2879 M in HBSS (PanEco, Russia) buffered with 0.01 M HEPES (PanEco, Russia) was added into the analytical chamber at a final concentration of 5 × 10⁷ cells/mL, pipetted, and the change in the DFL signal was recorded for 30 min. For negative control NGs on the cantilever were fixed by 99.8% methanol (Sigma-Aldrich, United States) for 20 min then cantilever deflections were studied in the system with bacteria.
Immediately after the experiment the NGs on the cantilever were fixed and studied using scanning electron microscope JSM-IT300LV (JEOL, Japan). The procedures of fixation and studying described in paragraph 2.1.6.

For immunophenotyping of SCs, the MSC Phenotyping Kit (130-095-198, Miltenyi Biotec GmbH, Germany) and antibody to CD31 (303124, Biolegend, United States) with corresponding control (400158, Biolegend, United States) were used according to the manufacturer’s instructions. Cells were washed with Versene solution (Paneco, Russia), detached from plastic with 0.05% trypsin-EDTA solution (Gibco, United States), and after trypsin inhibition by complete growth medium, they were aliquoted in four test tubes and centrifuged for 10 min at 300 g. Pellets were then resuspended in flow cytometry buffer [1% bovine serum albumin (BSA; Sigma, United States) in HBSS (Paneco, Russia)] and incubated for 10 min at 4°C with antibodies to CD31 or cocktail of antibodies to CD73, CD90, CD105, CD14, CD20, CD34, CD45 from the kit or corresponding control antibodies at a dilution provided by the manufacturer. After labeling, cells were diluted by 1 ml of flow cytometry buffer, centrifuged for 10 min at 300 g, then supernatant was discarded, and cells were resuspended in 500 μl of flow cytometry buffer. Cells were detected using BD LSR Fortessa flow cytometer (BD Biosciences, United States), and results were analyzed using the FlowJo software (BD, United States).