Alexa fluor 488 labeling kit

RNase3, RNase6, and RNase7 were labeled with Alexa Fluor 488 Labeling kit (Invitrogen, A10235), following the manufacturer′s instruction as previously described (21 (link)). To 0.5 mL of 2 mg/mL protein solution in PBS, 50 μL of 1 M sodium bicarbonate, pH 8.3, was added. The protein was incubated for 1 h at room temperature with the reactive dye, with stirring, and the labeled protein was separated from the free dye by PD-10 desalting column (GE Healthcare, 17-0851-01). Labeled protein distribution in cell cultures was followed by confocal microscopy. About 2.5 × 10⁵ RAW cells were harvested in 3 cm diameter microscopy plates (MatTek, P35G-1.5-14-C) 2–3 h before the assay. Macrophages were washed with RPMI and labeled with Hoescht 33342 (Thermo Fisher Scientific, 62249) and Cell Mask Deep Red Plasma membrane Stain (Thermo Fisher Scientific, C10046) at 0.5 μg/mL for 5–10 min before observation in Leica TCS SP5 AOBS equipped with a PL APO 63 × 1.4–0.6 CS oil immersion objective (Leica Microsystems, Mannheim, Germany). Following, Alexa Fluor labeled proteins were added at 2 μM to the cultures and time lapse was recorded at intervals of 30 s for 30 min. Fluorochromes were excited by 405 nm (Hoechst 33342), 649 nm (CellMask Deep Red), and 488 nm (Alexa Fluor 488). Emissions were collected with a HyD detector.

The following materials were purchased from commercial sources: Clear Immuno 384-Well Plate (Thermo Scientific, 8755), an anti-human IgG antibody (Bethyl Laboratories, A80-319A), Streptavidin-PolyHRP80 (Stereospecific Detection Technologies, SP80D50), Bovine Serum Albumin (BSA) for ELISA (Sigma-Aldrich, A7030), TMB Substrate (Surmodics, TMBS-1000–01), Carbonate-Bicarbonate Buffer capsule (Sigma-Aldrich, C3041), 96-well cell culture plate (Costar, 3799), BSA for cell assay (Sigma-Aldrich, A9418), Fetal bovine serum (FBS) (Sciencell, 0500), anti-mouse CD45 antibody-VioBlue (Miltenyi, 130-110-664), anti-mouse CD146 antibody-FITC (Miltenyi, 130-102-230), anti-human CD31 antibody-Pacific Blue (BioLegend, 303114), anti-non human primate CD45 antibody-APC (Miltenyi, 130-091-900), anti-CD32B (FcγRIIB) antibody (Sino Biological, 90014-R046), Liver Perfusion Medium (Gibco, 17701-038), Endothelial cell medium (Sciencell, 1001), Hepatocyte Culture Medium (Lonza, CC-3198), Alexa Fluor 488 Labeling Kit (Invitrogen, A20181), Alexa Fluor 647 Labeling Kit (Invitrogen, A20173). Other reagents were purchased from local commercial sources.

All plasmids and primers used in this study are summarized in Supplementary Tables 1 and 2, respectively. All restriction endonucleases, T4 DNA ligase, and Vent polymerase were purchased from New England Biolabs (Ipswich, MA, USA). The Taq polymerase and oligonucleotide primers were from Biosesang (Seongnam, Republic of Korea) and Cosmogenetech (Seoul, Republic of Korea), respectively. Difco^TM Terrific Broth, Ni-NTA agarose, Protein A agarose, and glutathione agarose 4B were obtained from Becton Dickinson Diagnostic Systems (Sparks, MD, USA), Qiagen (Hilden, Germany), Genscript (Scotch Plains, NJ, USA), and Incospharm (Daejeon, Republic of Korea), respectively. An Alexa Fluor 488 labeling kit, 1-Step Ultra-TMB substrate solution, GIBCO FreeStyle™ 293 expression medium, and sheep anti-hC1q-HRP conjugate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Polyethyleneimine-Max and goat anti-GST-HRP conjugate were obtained from Polysciences (Taipei, Taiwan) and GE Healthcare (Piscataway, NJ, USA), respectively. Unless stated otherwise, all other biochemical reagents were purchased from Sigma‒Aldrich (St. Louis, MO, USA).