Poly l lysine (pll)

Cells were seeded on glass coverslips coated with poly-L-lysine (Solarbio). After 48 h, cells were washed with ice-cold PBS. Next, 4% paraformaldehyde (Sigma) in PBS was used to fix cells for 30 min at room temperature, after which the cells were permeabilized by 10-min treatment with 0.4% NP-40. The cells were blocked with PBS containing 3% bovine serum albumin (ANRESCO) for 30 min at room temperature, and subsequently incubated with anti-γH2AX antibody (Millipore, 05616) or rabbit anti-Rad51 antibody (Abcam, Ab63801) at 4 °C overnight. The cells were then washed by using PBS buffer, and then incubated with Alexa 488 secondary antibodies (Invitrogen) and DAPI (Sigma-Aldrich) for 30 min at room temperature in the dark. The cells were washed with PBS for 3 times. Immunofluorescence images were analysed by the Zeiss780 Confocal Microscope.

The Seahorse sensor probe plate was hydrated with XF calibrant 10 h before the test. The 24-well cell culture plates were coated with Poly-l-lysine (50 mg/ml, Solarbio, China) according to manufacturer’s instructions. 200,000 cells were plated in 200 ml XF Seahorse medium (pH 7.4, Agilent, USA) supplemented with 2 mM glutamine, 10 mM glucose and 1 mM sodium pyruvate. Plates were centrifuged at 200 g without break and cells were then incubated for 30 min at 37 ℃ without CO₂. Afterwards, 300 ml of the same XF medium was added to each well. OCR was measured at basal conditions and after sequential stimulation of the cells by 1 mM Oligomycin (Oligo), 1 mM carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) and 0.5 mM Rotenone/Antimycin (Rot/AA) (all included in the Mitostress kit, Agilent, USA). After the same pre-treatment, glycoPER was measured under basal conditions and with the sequential addition of 0.5 μM Rot/AA and 50 mM 2-deoxy-D-glucose (2-DG) (all included in the Glycolytic rate kit, Agilent, USA).

Live-cell imaging was performed as described (85 (link)). Briefly, after synchronization with 5% sorbitol at the early ring stage (3 hpi), parasites were allowed to mature; schizonts (42 hpi) were purified from uninfected RBCs using 40%/70% Percoll as described above and treated with 10 μM E64 cysteine protease inhibitor (ThermoFisher) for 3 to 4 h to prevent egress; and the parasites were incubated with fresh RBCs at 2% hematocrit in RPMI complete medium containing dyes from the Image-IT live plasma membrane and nuclear labeling kit (Thermofisher) at 37°C for 10 min. The labeled parasites were washed twice in PBS and then plated on a cell imaging cover glass (Eppendorf, Hamburg, Germany) coated with poly-l-lysine (Solarbio, Beijing, China). Live-cell imaging of the parasites was performed on a Leica Stellaris 5 confocal microscope. The sample chamber was maintained at 37°C and supplied with a humidified atmosphere under 5% CO₂.

Costar culture plates (Corning Inc) were coated with 100 µg/mL poly‐L‐lysine (Solarbio) according to manufacturer's instructions before freshly isolated neutrophils (1 × 10⁷ cells/mL) were gently added. After incubation at 37°C in 5% CO₂ for 0.5‐1 hours, neutrophils were stimulated with APS‐IgG (15 µg/mL), HC‐IgG (15 µg/mL) and phorbol‐12‐myristate‐13‐acetate (PMA; 50 nmol/L; Sigma‐Aldrich, St. Louis, MO) or left untreated.

U87MG and A172 cells (1 × 10³/well) were seeded into 6-well plates, and P3 cells were seeded into 6-well plates coated with poly-l-lysine (Solarbio). After 2 weeks, cells were fixed with 4% paraformaldehyde and stained with crystal violet (Solarbio). The total number of colonies was counted per well. All experiments were performed in triplicate.

The freshly cleaved mica was cleaned with 3% hydrochloric acid with 96% ethanol for 20 times. Afterward, they were coated for 1 h in a 0.001% poly‐l‐lysine (Solarbio) solution, rinsed with Milli‐Q water (18.2 MΩ.cm), and dried overnight at room temperature. The poly‐l‐lysine modified mica was stored at 4 °C for no more than one month before use. The solution of sEVs was diluted ten times with PBS, filtered with a 0.22 µm filter (Millipore, USA), deposited on the poly‐l‐lysine modified mica for absorbing sEVs and gently rinsed with PBS before imaging. AFM images were acquired in liquid under PeakForce QNM mode on Dimension FastScan AFM (Bruker, Billerica, CA) under the bio model (also called FastScan Bio AFM) using MLCT‐A probes (silicon nitride tip, nominal force constants 0.07 N m⁻¹, nominal radius 20 nm, resonant frequency 15–30 kHz, Bruker, US). The imaging force was preset to be 0.5–3 nN. The force‐indentation curves were obtained at an indentation velocity of 250 nm s⁻¹ until the surface was reached. The force‐indentation curves of the AFM tip approaching the mica surface were checked before and after each imaging of sEVs to make sure no contaminations from the sEVs adhered on the tip.

SYTOX green acid staining was performed to assess pyroptosis induced by OGD/R and PM2.5 exposure as described before.³² Microplates were treated with 0.05 mg/mL poly‐L‐lysine (Solarbio Life Sciences, Beijing, China) before HMC‐3 cells were seeded. Then, the cells were seeded in the 96‐well plates and SYTOX green acid staining solution was prepared by diluting stock solution (Invitrogen, Carlsbad, CA, USA) at 1:30000 (167 nM) in phosphate‐free buffer. SYTOX and DAPI were added to cover the cells for 15 min in the dark. The cells were then washed three times in phosphate‐free buffer and quickly imaged using a confocal microscope (Leica, Wetzlar, Germany).