Facsymphony a3

The effect of IPS-2 treatments on PC-3 cell death stages were studied using the apoptosis assay kit (Biovision Inc., Milpitas, CA, USA) (Annexin V FITC; and PI), as reported earlier [54 (link)]. In brief, the PC-3 cells were seeded in the T-25 flask (RPMI medium) and grown in humidified CO₂ (5%) atmosphere at 37 °C. After reaching adequate confluence (75–85%), the cells were treated with IPS-2 (in 250 and 500 µg/mL of working concentration, separately) and incubated for 18 h. Following incubation, the cells were collected, washed twice with ice cold PBS (pH 7.4) through centrifugation (2000 rpm for 5 min at 4 °C), and dispersed in Annexin binding buffer (1X,100 µL). Then, 1 µL of PI (100 µg/mL) and 5 µL of FITC (as provided in the kit) were added and incubated for 15 min at room temperature. Later, the final volume was made up by adding 400 µL of Annexin binding buffer. Finally, the cell death stages were quantified by reading the cells using a flow cytometry analyzer-II (FACSymphony A3; BD, Franklin Lakes, NJ, USA).

For T cell activation assay, lymph nodes (superficial cervical, axillary, brachial, inguinal) and spleen from C57BL/6 wild-type mice or Gpr52^–/– and respective littermate controls were collected in ice-cold PBS. CD4+ T cells were isolated from single-cell suspension using the MojoSort CD4+ T cell isolation kit (BioLegend) according to the manufacturer’s protocol. CD4+ T cells were seeded in an anti-CD3 (1 µg/ml; clone 145-2CL11, BioLegend)-coated 96-well plate. Cells were supplemented with compounds (E7 and FTBMT) and soluble anti-CD28 (2 µg/ml; clone 37.51, BioLegend). Samples were incubated for 6 hours at 37°C and 5% CO₂. After that cells were stained for T cell surface markers for 30 minutes at 4°C. Dead cells were excluded by adding Alexa Fluor 750 NHS Ester (Succinimidyl Ester) (Thermo Fisher). Nonspecific Fc receptor-mediated antibody binding was minimized by blocking with TruStain FCX anti-mouse CD16/32 (clone 93, BioLegend). After fixation and permeabilization at RT with eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher), FOXP3 was stained intranuclearly for 30 min at RT. Cells were stained with antibodies listed in Supplementary Table 1. Samples were acquired on the BD FACS LSR II analyzer (BD Bioscience) or FACSymphony A3 (BD Bioscience). Data were analyzed using FlowJo (version 10, BD Bioscience, RRID: SCR_008520).

¹H NMR spectrum were performed on a NEO 400 MHz spectrometer. LC-MS was performed on a Waters Acquity LCMS system equipped with UV-Vis and MS detectors. Flash chromatography was conducted on a Teledyne Isco CombiFlash Rf-200i chromatography system equipped with UV-Vis and evaporative light scattering detectors (ELSD). Particle size and zeta potential were measured by dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS. Particle morphology was measured by Cryo-TEM. Flow cytometry was performed using a FACSCanto or FACSymphony A3 instrument (BD Biosciences). In vitro luminescence intensity, pKa, encapsulation efficiency and mRNA concentration, and cell viability were quantified using an Infinite M Plex plate reader (Tecan, Morrisville, NC).

Cryopreserved PBMC were thawed and counted with Trypan blue and then stained to determine the different cellular subsets, according to standard staining procedures. First, to exclude dead cells from analyses PBMC were stained with eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific, Waltham, MA USA), according to the manufacturer’s instructions. Then, PBMC subsets were identified by staining with pre-determined dilutions of fluorochrome-conjugated anti-human antibodies for 20 min at 4 °C (See Supplementary Tables 3, Additional File 3). For markers which needed intracellular staining, the Fixation/Permeabilization Solution Kit (BD) was used. Stain buffer used for washing and antibodies incubations consisted of 0.02% sodium azide and 0.1% bovine serum albumin in PBS (Sigma-Aldrich). Cells were acquired on FACSAria III or FACSymphony A3 calibrated with CST beads before each experiment (all from BD Biosciences). Electronic compensation was performed using single-stained controls prepared with control PBMC and antibody-capture beads (BD Biosciences). Data were acquired with the FACSDiva V 8.0 software (BD) and analyzed using FlowJo 10.8 software (Treestar, LLC, USA).

Freshly isolated lymphocytes from livers and lungs were restimulated with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (500 ng/mL) for 4 h in the presence of monensin (2 µM) and then stained for ILC2 surface markers. The cells were fixed with 4% PFA for 10 min at RT and permeabilized in permeabilization buffer (50 mM NaCl, 5 mM EDTA, 0.02% NaN₃, pH 7.5) containing 0.5% Triton X-100 for 10 min on ice. After blocking with 3% bovine serum albumin in PBS for 15 min, the cells were stained on ice for 30 min with anti-IL-13 (BioLegend, 159403, W17010B, 1:100). Flow cytometry was performed with a FACSCanto II (BD Biosciences) or FACSymphony A3 (BD Biosciences) flow cytometer, and the results were analyzed with FlowJo.

The antioxidant effect of BFR-EVs was evaluated by H2DCFDA staining and qRT-PCR analysis. Briefly, HDFs were seeded into 6-well plates (SPL) at a density of 1.5 × 10⁴ cells/cm² and cultured at 37 °C in a humidified atmosphere with 5% CO₂ for 24 h. HDFs were then treated with different concentrations of BFR-EVs in serum-free DMEM/F12 for 24 h. Intracellular ROS were induced by H₂O₂ treatment and UVB (315 nm) irradiation. To generate H₂O₂ induced ROS, cells were washed with PBS several times and treated with 0.5 mM H₂O₂ for 3 h. After 3 h of H₂O₂ treatment, a specific assay was performed. For UVB-induced ROS generation, cells were washed with PBS several times and 700 μL of PBS was added to each well. Cells were then irradiated with 80 mJ/cm² of UVB generated by a Bio-Sun illuminator (Vilber Lourmat, Eberhardzell, Germany). After UV irradiation, cells were maintained in serum-free DMEM/F12 for 24 h before a specific assay. Specific analysis was then performed after washing the cells several times with PBS. Intracellular ROS levels were observed by H2DCFDA staining. Cells were treated with 10 μM H2DCFDA for 30 min. Cell nuclei were stained with 2.5 μg/mL Hoechst 33342 for 20 min. Subsequently, the cells were washed three times with PBS. Intracellular ROS levels were analyzed using a fluorescence microscope and a flow cytometer (FACSymphony™ A3, BD, San Jose, CA, USA).

All antibodies for flow cytometry were purchased from BD Biosciences, BioLegend, Thermo Fisher Scientific, Cell Signaling Technology and R&D Systems. D^bGP33–41 and D^bGP276–286 tetramers were prepared in house and were used to detect LCMV-specific CD8⁺ T cells. Streptavidin-PE or streptavidin-APC was purchased from Thermo Fisher Scientific. Dead cells were excluded by using the Live/Dead Fixable Near-IR or Yellow Dead Cell Stain kit (Thermo Fisher Scientific). For cell-surface staining, antibodies were added to cells at dilutions of 1:20 to 1:500 in PBS supplemented with 2% FBS and 0.1% sodium azide for 30 min on ice. Cells were washed three times and fixed with 2% paraformaldehyde. To detect cytokine production, 1 × 10⁶ splenocytes were stimulated with a pool of nine LCMV-specific peptides (200 ng ml^–1 each of GP33–41, GP70–77, GP92–101, GP118–125, GP276–286, NP166–175, NP205–212, NP235–249 and NP396–404) in a 96-well round-bottom plate for 5 h at 37 °C in a CO₂ incubator in the presence of GolgiPlug (BD Biosciences). Samples were acquired on a Canto II, LSR II or FACSymphony A3 instrument (BD Biosciences) with FACSDiva (v9.1; BD Biosciences), and data were analysed by using FlowJo (v9.9.6 or v10.8.1; BD Biosciences).