Bp151 100

The frozen sections were fixed with 4% paraformaldehyde (Sigma #P6148) and permeabilized with 0.25% Triton X-100 (Fisher Scientific #BP151–100). After a wash with PBS/Tween-20 (Fisher Scientific #BP337–100), slides were treated with 3 M hydrochloric acid (Fisher Scientific #A144S-500) for 10 min to open the nucleus structure if needed. Slides were blocked with 10% donkey serum (Sigma #D9663) in 2% BSA (Sigma) in PBS/Tw-20 for 1 h at room temperature. Afterwards, slides were probed with primary antibodies (CD31 1:25, BD #550274; BrdU-APC 1:50, BD; Alexa Fluor 488 Mouse anti-BrdU 1:10, BD #558599; Flag M2 1:500, Sigma #F1804) and incubated overnight at 4 °C. The next day, slides were washed and incubated with the fluorescence-conjugated secondary antibody (AF488 donkey anti-rat 1:300, Invitrogen #A-21208; AF594 donkey anti-mouse 1:300, Invitrogen #A-21203; AF594 donkey anti-rat 1:300, Invitrogen #A-21209). Images were taken with a confocal microscope LSM880 (Zeiss) and analyzed by Zen software (Zeiss).

Murine peritoneal macrophages RAW264.7 (ATCC TIB-71) were grown in Dulbecco’s Modified Eagle Media (DMEM) (Thermo Scientific, Waltham, MA, USA) supplemented with 10% (v/v) heat inactivated fetal bovine serum (JRScientific, Woodland, CA, USA), HEPES (10 mM), and glutamine (2 mM) in a 37 °C incubator with a 5% CO₂ atmosphere. Prior to infections, cells were seeded in a 24-well tissue culture plate and grown for 14–18 h. Overnight cultures of L. monocytogenes were used for infections at a MOI of 10. Bacteria diluted in cell culture medium were added to each well (500 µL) and incubated for 30 min. Following incubation, media were aspirated, and cells were washed twice with sterile Dulbecco’s phosphate-buffered saline (DPBS). Fresh media (1 mL per well) containing 10 µg/mL gentamicin stock was added to each well. To enumerate intracellular bacteria, cell culture media were aspirated off and sterile 0.1% (v/v) Triton X-100 (Fisher BP151-100) was added to each well (200 µL per well) to lyse host cells. Host cells were lysed at 1, 2, 4, and 8 h post infection (hpi). Lysates were diluted and spread on LB plates. Colonies on plates were counted using an automatic colony counter (Synbiosis aCOLyte 3) after 24–48 h of incubation in a 37 °C incubator.

Following the EV treatment, on day 5 of culture, TG neurons were washed once with 1 × PBS and fixed in 4% paraformaldehyde (PFA, Ted Pella, #18505) for 15 min. Samples were washed three times with 1 × PBS for 5 min each with gentle agitation. To permeabilize the cell membrane, samples were incubated in 1 × PBS + 0.1% Triton-X-100 (Fisher, #BP151-100) for 1 h at room temperature (RT). Next, samples were incubated in a blocking buffer containing 1 × PBS, 0.05% Tween-20, and 1% BSA for 1 h at RT and then incubated in the dark with a FITC-conjugated antibody against B3 tubulin (Biolegend, #801203) at a concentration of 1:300 dilution for 90 min at RT. Cells were then washed with PBST (PBS + 0.05% Tween-20) three times for 10 min each with gentle agitation. Following washes, samples were incubated with nuclear stain Hoechst 33,342 (in a 1:5000 dilution) for 5 min. After incubation, samples were washed three times with 1 × PBS for 10 min and stored in 1 × PBS for confocal imaging (Zeiss, LSM 800).

Forty-thousand iCMs were seeded in one well of an eight-chamber slide (#155409 Lab-TekII, Thermo Fisher Scientific). Cells were fixed with 4% PFA in PBS, and blocked with 5% FBS, 0.3% Triton-X (#BP151-100, Fisher Scientific, Waltham, MA, USA) and 1% bovine serum albumin (#SLCB8319, Sigma-Aldrich) in PBS (#21030, Corning) overnight. Cells were stained with mouse anti-cardiac troponin (Gladka et al., 2021 (link)) used at 1:400 dilution (ab8295, Abcam, Waltham, MA, USA). Secondary antibody was goat anti-mouse Alexa Fluor 488 used at 1:1000 dilution (#A11001, Invitrogen). Cells were washed twice with PBS, and one drop of propidium iodide (#R37108, Molecular Probes, Invitrogen) was added. The slide was treated with Anti-Fade Fluorescence Mounting Medium (ab104135, Abcam) and imaged with a Zeiss fluorescent microscope (Zeiss AxioVision 4.8 software).

Patient peripheral blood was collected in heparinized vacutainer tubes (BD Biosciences, Franklin Lakes, NJ, USA) and diluted 1:2 with phosphate-buffered saline (PBS; 1.37 M NaCl, 27 mM KCl, 0.1 M Sodium phosphate di basic, 18 mM KH2PO4, pH 7.4) solution. Peripheral blood mononuclear cells (PBMCs) were isolated using density centrifugation with Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA) as previously published [9 (link)]. Ficoll (12 mL) was layered underneath diluted blood, then centrifuged for 20 min at 800× g with no brake. Isolated PBMCs were resuspended in buffer, then adhered to poly-D-lysine (1 mg/mL) coated slides (Millipore, Burlington, MA, USA; Fisher Scientific, Waltham, MA, USA) at 37 °C for 15 min. Cells were fixed with 4% PFA for 5 min, permeabilized with 0.5% Triton-X (Fisher Scientific, BP151-100) for 10 min, and fixed again with 4% PFA for 10 min.

Washed Red Blood Cells (RBCs) were purchased from (Innovative Research Inc, Catalogue#: IWB3ALS40ML). 1 mL of RBCs was washed once in cold 1X PBS and then resuspended in 20 mL of cold 1X PBS. In a 96-well format, 10 µL of RBCs was added to each well containing 90 µL of either peptide pre-diluted in 1X PBS, 1X PBS only, or 1X PBS + 0.1% Triton-X100 (Fisher Scientific, Catalogue#: BP151-100). Cells were briefly mixed on an orbital shaker then incubated at 37 °C for 1 h with 5% CO₂. After incubation, cells were pelleted in a benchtop centrifuge at 500 rcf for 3 min and supernatants were collected for reading at 490 nm on a Biotek Synergy 2 Multi-Mode Microplate Reader. The percentage of RBC lysis was calculated by linear regression using the PBS and PBS + 0.1% TritonX-100 as 0% and 100% lysis controls, respectively. Statistical significance was calculated by two-way ANOVA with Bonferroni’s correction.