Nucblue hoechst 33342

ActinRed^TM 555, ActinGreen^TM 488, NucBlue Hoechst 33342 were from Molecular Probes (Oregon, OR, USA). AnnexinV-FITC was from BD bioscience (San Jose, CA, USA). DRAQ7^TM was from Biostatus (Shepshed, UK). Trichostatin A (TSA) was from Sigma-Aldrich (Steinheim, Germany). May-Grünwald Eosine-methylene blue solution (product number: HX68862424) and Giemsa Azur-Eosine-methylene blue solution (product number: HX128350) were from Merck KGaA (Darmstadt, Germany). SYBR GreenER SuperMix and Rox reference dye were from Invitrogen (Carlsbad, CA, USA).

Rabbit polyclonal anti-histone H2A, H2B, H3 and H4, mouse monoclonal anti-tryptase [AA1], rabbit monoclonal anti-lamin B1, rabbit polyclonal anti-hnRNP A2B1 antibodies were from Abcam (Cambridge, UK). Rabbit polyclonal anti-histone H1 antibody and PrestoBlue^TM cell viability reagent were from Invitrogen (Carlsbad, CA). Goat polyclonal to β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antisera to mMCP6 and CPA3 were as described^{49 (link)}. Recombinant human skin β-tryptase and human lung β-tryptase were from Promega (Madison, WI) or prepared as described^{39 (link)}. Recombinant human hnRNP A2B1 was from Abcam. Click-iT^TM Plus EdU Pacific Blue^TM flow cytometry cell proliferation kit, ActinRed^TM 555, ActinGreen^TM 488, NucBlue Hoechst 33342 and wheat germ agglutinin Alexa^TM 488 were from Molecular Probes (Oregon, OR). AnnexinV-FITC was from BD bioscience (San Jose, CA). DRAQ7^TM was from Biostatus (Shepshed, UK). AC 55541 was from Tocris bioscience (Bristol, UK). Dynasore hydrate, Nafamostat mesylate, Heparin sodium salt and Trypan Blue solution were from Sigma-Aldrich (Steinheim, Germany).

Lysate (125 μg/cm²) or 10% FBS (∼1,600 μg/cm²) were used to pre-condition the surfaces of TCPS 96 well plates for 60 min, then seeded with RAW-Blue reporter macrophages (1.5 × 10⁵ cells/cm²; DMEM supplemented with 10% FBS, 5 μg/ml plasmocin and 200 μg/ml zeocin) and incubated for 72 h at 37°C and 5% CO₂.
After 72 h, cell morphology and quantification of fusion and multinucleated cell size was assessed using brightfield and fluorescence microscopy. The Raw-Blue™ macrophages were incubated for 20 min at 37°C with staining solution containing 2 drops/ml Hoechst 33342 (NucBlue, ThermoFisher Scientific; Ex/Em: 360/460 nm) and 10 μg/ml Wheat Germ Agglutinin (WGA) Alexa Fluor™ 488 conjugate (ThermoFisher Scientific; Ex/Em: 495/519 nm) to demarcate the nucleus and plasma membrane, respectively. Labeled macrophages were imaged at 40x using a Nikon Eclipse Ti2 microscope, using the NIS Elements Advanced Research imaging software. Three fields of view were randomly selected near the center area of each well. Nuclei enumeration (Hoechst) and size measurements of multinucleated cells (WGA-labeled area) was performed in ImageJ (Schneider et al., 2012 (link)). FBGC were classified as cells containing three or more nuclei to exclude cells undergoing mitosis (Saleh et al., 2020 (link)). The percent fusion was quantified as $\text{\% fusion}=\frac{\text{number of nuclei in FBGCs}}{\text{total number of nuclei}}\times 100\text{\%}$