Dylight 650

Immortalized NPCs (1 × 10⁶ cells) supplemented with 1 × 10⁴ hiPSCs (1%) were seeded onto hESC-qualified Matrigel-coated 6-well plates in mTeSR Plus, and hiPSC-NPC preparations (2.5 × 10⁶ cells) supplemented with 2.5 × 10⁴, 250, and 50 hiPSCs (1%, 0.01%, and 0.002%, respectively) were seeded onto hESC-qualified Matrigel-coated 60-mm dishes in STEMdiff neural progenitor medium (Veritas) with 10 µM Y27632 (Fujifilm Wako Pure Chemical Corporation). Cells were transduced with the Ad vector at 3 or 18 pfu/cell for 24 h, followed by adding 10 nM AP1903. Twenty-four hours after treatment, cells were harvested by treatment with Accutase, and the cell suspension was incubated with an anti-TRA-1-60 monoclonal antibody conjugated with DyLight 650 (Thermo Fisher Scientific, #MA1-023-D650; 1:100) and with a FITC conjugated rBC2LCN (Fujifilm Wako Pure Chemical Corporation, #186-02,993; 1:100) on ice for 1 h, followed by washing with PBS. In the experiments using hiPSC-NPC preparations, the cells were fixed using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) on ice for 30 min. The stained cells were determined and analyzed using a cytoFLEX S (Beckman Coulter, Brea, CA, USA). The measurements were stopped when 2 × 10⁴ cells in the gates 1 and 2 shown in Supplementary Figs. S3, S8, and S9 were counted.

All DNA below 90 bp were purchased from Integrated DNA Technologies, and DNA of greater length was synthesized by PCR. FAM-labelled DNA was also purchased from Integrated DNA Technologies. DyLight-550 and DyLight-650 maleimides were purchased from Thermo.

Primary antibodies used were: chicken anti-LTBP-2 polyclonal (1:100, made by immunizing chickens with recombinant mouse LTBP-2, Figs 5 and 6, Supplementary Figs S6, S7 and S8), goat anti-LTBP-4 polyclonal (1:100; R&D Systems, Figs 5 and 6, Supplementary Figs S6, S7 and S8), rabbit anti-mouse fibrillin-1 polyclonal (1:100, previously described13 (link), Figs 4–6, Supplementary Fig. S6), rabbit anti-elastin polyclonal PR385 (1:100; Elastin Products Company), and mouse anti-fibulin-5 monoclonal 10A (1:100, previously described26 (link), Supplementary Fig. S8). Secondary antibodies conjugated with Alexa 488, 546 and Dylight 650 were purchased from Thermo Fisher Scientific (Alexa 488 and 546) and Leinco Technologies (Dylight 650). Frozen sections were fixed with acetone at −20 °C for 5 min, blocked with PBS containing 1% bovine serum albumin and 0.05% Triton X-100 before immunofluorescent staining. Immunofluorescence images were obtained using all-in-one fluorescence microscope BZ-9000 (Keyence).

Following the protocol outlined in [33 (link)], 2x10^6 adherent, IL-8 treated neutrophils were synchronously infected with 2x10^6 Tag-It Violet stained Gc by centrifugation (MOI 1). After incubation for the indicated times at 37°C with 5% CO₂, paraformaldehyde was added (final concentration 2%) and cells were lifted with a cell scraper (Corning 353085). Cells were washed in PBS and blocked in 10% normal goat serum. Extracellular associated bacteria identified by reaction with rabbit anti-Gc antibody (Meridian B65111R) that was conjugated in-house with 1 μg/mL Dylight 650 (Thermo Scientific) according to manufacturer’s recommendations. Results are expressed as the percentage of neutrophils with at least 1 associated (bound and/or internalized; Tag-IT Violet+ Dylight 650+) Gc and the percentage of neutrophils with at least 1 internalized (Tag-IT Violet+ Dylight 650-) Gc.