Facscanto a

Artificial cells were diluted in either 300 mM alanine or PBS before analysis. Flow cytometry was with a FACSCanto A (BD Biosciences) with a flow rate setting of “low” and voltage values of 350 V (for SSC-W), 400 V [SSC-A, FSC-A, and FITC (fluorescein isothiocyanate) for Alexa Fluor 488–dextran and pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid)], or 700 V (FITC for GFP and sfGFP). For each run, at least 10,000 gated events or 50,000 total events were recorded with a gating strategy that took into account only fractions that contained giant vesicles (fig. S3D) (53 (link), 54 (link)). The flow cytometer was calibrated with 1- and 10-μm particles for voltage optimization and particle size determination. Single and double events were discriminated by SSC-A versus SSC-W plots, and only single events were taken into account for statistical analyses (fig. S8B). For the experimental gating strategy, the “No DNA” and/or “No dextran/GFP” samples were used as negative controls to determine the FITC-positive events to create a new gate. Both the loss of FITC intensity (due to GFP escape through PFO pores) and percent changes in events of specific gates were taken into account for statistical analysis. The data were analyzed with either FACSDiva software (BD Biosciences) or FlowJo v10 (FlowJo LLC). All events were plotted in pseudocolor.

Fresh cell suspensions were prepared from macaque spleen, axillary and inguinal LNs. Peripheral blood was collected to EDTA-coated tubes. Fresh cells were stained with a panel of monoclonal antibodies. The fluorochrome-conjugated antibodies used are provided in the S2 Table. Intracellular Bcl-6, c-Maf, Foxo1, KLF2, Eomes, and T-bet staining was performed after fixing and permeabilizing the cells with the FoxP3 staining buffer set (eBioscience). After lysing erythrocytes (Lysing buffer Pharm Lyse 10X BD Biosciences), sixty thousand events corresponding to mononuclear cells were recorded in FACS Canto A (BD Bioscience). Analyses were performed using FlowJo software (Tree Star, Inc.).

The effect of AuNP-PEG-FA on cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega). Briefly, 1 × 10⁴ B16F10 cells were seeded in 96-well plates and incubated at 37 °C, 5% CO₂. After 24 h, the medium was replaced with 100 μL of increasing concentrations of AuNP-PEG-FA in RPMI medium and incubated for another 24 h. The cell viability was measured (in quintuplicate) in three independent experiments using the MTS assay according to the manufacturer’s protocol. Cell death by apoptosis or necrosis was evaluated before EV isolation by flow cytometry. Briefly, B16F10 cells were grown to 50% confluency and incubated with AuNP-PEG-FA (1 nM) for 6 h at 37 °C, 5% CO₂. Non-incorporated nanoparticles were discarded by washing 3 times with PBS and the medium was replaced with RPMI supplemented with 10% of EV-free serum prepared as previously described [64 ]. After 24 h, cells were harvested and marked with FITC Annexin V and propidium iodide (PI) using a cell death/apoptosis kit (Invitrogen), according to the manufacturer's protocol and analyzed by flow cytometry with a FACScanto A (BD Biosciences).

The experiments used CHO-CR3 cells transduced to express human CD11b (CHO-CR3 CD11b⁺ cells). CHO-CR3 CD11b⁺ cells were incubated for 30 min on ice in binding buffer with 40 nM biotinylated CyaA-HPV16 E7 and 0 to 512 nM CyaA, 0 to 512 nM test substance, or 512 nM heat-inactivated GTL001. After washing, cells were incubated for 30 min on ice with 0.5 mg/mL PE-labeled streptavidin. Cells were re-suspended in 10 nM TO-PRO-3. Bound biotin-CyaA-HPV16 E7 was detected by flow cytometry using a FACSCanto A equipped with a high-throughput sampler (BD Biosciences). Analyses were performed after gating on living cells as based on exclusion of TO-PRO-3 positive cells. The dose-response curves were fitted using Prism (GraphPad Software, La Jolla, CA, USA) to estimate the concentration resulting in 50% inhibition (IC₅₀).

Whole blood (100 μl) was stained for Ly-6G (neutrophils, DCs) (clone 1A8, BD Bioscience), CD14 (monocytes) (clone rmC5–3, BD Bioscience), and F4–80 (monocytes) (clone BM8, Biolegend) to quantitate granulocytes. AccuCount Fluorescent Particles (12.5 μl of 5.2 μm size, Spherotech, Inc.) were added to calculate absolute counts (cells/μl). Whole blood was lysed after staining with FACS lysis buffer (BD Bioscience) and analyzed on a FACS Canto A (BD Bioscience) with DiVa Software and further analyzed using FlowJo Analysis Software (Tree Star Inc.). Cells were gated for forward and side scatter and dead cells (a very small fraction) were excluded. The total numbers of granulocytes were quantified as: (the number of events (Ly-6G high, CD14-, and F4–80-) divided by (number of events for the AccuCount particles) times (number of AccuCount particles per 12.5 μL times volume of test sample used (0.1 ml)). In some samples, absolute counts of granulocytes were measured using volumetric/flow-rate calibration [35 (link),36 (link)]. Stained and lysed whole blood was analyzed for a fixed amount of time that resulted in identical amounts of volume analyzed. All samples from each experiment were analyzed at the same time to avoid possible variation in flow rates that could occur at different days, temperatures, or relative humidities.

HCT shNT or shDHX30 were seeded in 6-well plates at a concentration of 3 × 10⁵ cell/well. 48 h after seeding, the cells were trypsinized, washed with 1X PBS, and counted for subsequent staining according to BD Cycletest Plus DNA Kit protocol. Briefly, 5 × 10⁵ cells were centrifuged, the supernatant was removed, and 250 µL of Solution A were added. The cells were resuspended and incubated for 10 min before adding 200 µL of Solution B. After 10 min of incubation, 200 µL of ice-cold Solution C were added to the samples and the cells were incubated for 10 min at 4 °C in the dark before analysis with FACS CantoA (BD). The percentage of cells in each stage of the cell cycle (G1, S or G2) was computed using the ModFIT LT 4.0 software (Verity Software House).