Polybead carboxylate microspheres

Polybead carboxylate microspheres (unlabeled, nominally 0.05 mm; no. 15913; Polysciences, Warrington, PA, USA) were glycine coated, as described (25 (link)) and referred to as PS50G. To investigate the long-term effects of PS50G on the innate immune response, mice received saline or PS50G (200 µg/50 µl) intratracheally on day 0 and lymph nodes (LN) and lungs were collected on days 1, 3, 7, and 30 post instillation. In some experiments, 10 µg lipopolysaccharide derived from Escherichia coli (Sigma-Aldrich, St. Louis, MO, USA) were used as a positive “inflammatory” control. The effects of PS50G on acute allergic asthma were investigated by intratracheally instilled PS50G (200 µg/50 µl) into mice on days 0 and 2 prior to intraperitoneal sensitization with ovalbumin (OVA) (50 µg; Sigma-Aldrich, St. Louis, MO, USA) in aluminum hydroxide (General Chemical, Parsippany, NJ, USA) on days 12 and 22 and intranasal OVA challenge (25 µg) on days 32, 34, 37, and 39. Tissue sampling was performed 24 h after the final lung allergen challenge (day 40) as described (8 (link), 9 (link)).

HMC 1.2 or EoL-1 cells (5 × 10⁵/ml) were incubated for 1 h on ice with sCD30, ultracentrifugation-enriched CD30-containing EVs or HBSS buffer alone. After washing with PBS containing 1% albumin and 0.1% sodium azide, cells were incubated for another 30 min on ice with FITC-coupled SGN-35 (0.1 μg/ml). After washing in the above PBS, cells were evaluated by flow cytometry. Vesicles alone were incubated overnight with polybead carboxylate microspheres (4.5 μm; Polysciences INC, Warrington, PA). The beads were blocked with 1% BSA (v/w) in PBS. Then, aliquots were incubated with unlabeled or fluorescence-labeled antibodies. Aliquots with unlabeled antibody were in a second step labeled with fluorescence-labeled anti-mouse IgG (Dianova, Hamburg, Germany). Beads were evaluated by flow cytometry.

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and TexasRed® tagged 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (TexasRed® DHPE), were purchased from Avanti Polar Lipids (AL, USA) and Invitrogen (CA, USA), respectively. The GV membrane composition was DOPC: TexasRed® DHPE = 99.7:0.3 (mol%). Polybead® carboxylate microspheres (R = 1.00 μm; 2.5 vol%; 1.05 g/cm³) and Fluoresbrite® yellow green carboxylate microspheres (R = 1.00 μm; 2.5 vol%; 1.05 g/cm³; λex, 441 nm; λem, 486 nm) were purchased from Polysciences, Inc. (PA, USA). _D-Glucose, sucrose, and liquid paraffin were purchased from Wako Pure Chemical Industries (Osaka, Japan). The Tris-HCl buffer stock solution (100 mM, pH 7.5, 1.34 g/cm³) was prepared by adjusting pH of Tris (hydroxymethyl)-aminomethane (6.057 g, 50 mmol) aqueous solution to 7.5 with 1 M HCl.

For the generation of P-selectin-coupled beads polybead-carboxylate-microspheres (2 µm, 2.7% solids (w/v) Polysciences), polylink protein coupling kit (Polysciences), and recombinant human P-selectin protein (carrier free, Catalog no. ADP3-050, R&D Systems) were used. Coupling was performed according to the manufacturer’s protocol. About 3.1 mg microparticles were centrifuged (1000g, 10 minutes) resuspended in polylink coupling buffer twice before 21-mg carbodiimide was added for activation. After 15 minutes, 50-µg P-selectin was added and incubated for 120 minutes. Then, beads were centrifuged (1000g, 10 minutes) and washed in storage buffer twice and stored at 4 °C in 100-µL storage buffer. As controls uncoupled beads (without protein incubation) and L-selectin-coupled beads were generated using recombinant human L-selectin (R&D Systems).

Ibrutinib, acalabrutinib, entospletinib, idelalisib and dyngo-4A (SelleckChem), and cyctochalasin D (Merck) were dissolved in dimethyl sulfoxide (DMSO). The antibodies used were goat F(ab’)₂ anti-human IgM, control goat F(ab’)₂ and mouse F(ab’)₂ κ anti-IgM (all Cambridge Biosciences). These were used as soluble antibodies or after covalent binding to 2.8 μm epoxy-coated M-280 Dynabeads (ThermoFisher) or 3 μm carboxylate-coated latex beads (Polybead^® Carboxylate Microspheres; PolySciences) using the manufacturers’ instructions.