Nucblue live

Fluorescently labeled LNPs incorporating 0.1% Rhodamine/1,2-dioleoyl-sn-3-phosphatidylehanolamine (DOPE)^{14 (link)}, and encapsulating Firefly Luciferase (FLuc) reporter mRNA were used to transfect HeLa cells (4k cells per well) in 96-well plates at 10 ng mRNA per well. HeLa cells were stained with DAPI (NucBlue™ Live, ThermoFisher) and CellTracker™ Deep Red (ThermoFisher) prior to LNP dosing, and live-cell imaging was done every hour for 24 h on Opera Phenix high-throughput spinning disk confocal (Perkin Elmer) equipped with environmental control using a 20x water immersion objective (NA 1.0). Image analysis was done in Harmony (PerkinElmer), individual cells were segmented using DAPI and CellTracker channels, and LNP accumulation in endocytic vesicles was quantified using spot segmentation in the Rhodamine channel. LNP accumulation at the single-cell level was quantified as cytosolic spots intensity sum normalized to cell area and reported as average per well (three wells per sample, five fields of view, and approximately 1000 cells per well).

Neonatal (7 days after birth) cardiomyocytes were isolated as described previously (Mahmoud et al., 2014 (link)). Briefly, mice were intraperitoneally injected with 100 μL heparin (6.25 U/μL) to prevent clotting. Thirty minutes later, the mice were anesthetized with 2% isoflurane, and then dissected hearts were ligated to a Langendorff perfusion system through the aortic cannula. The hearts were firstly perfused with perfusion buffer (140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 10 mM HEPES, 10 mM taurine, 10 mM 2,3-butanedione monoxime and 10 mM glucose, pH 7.3) for 5 min to remove residual blood and secondly digested with digestion buffer (perfusion buffer containing 1 mg/mL collagenase II, 0.12 mg/mL trypsin and 0.02 mM CaCl₂) for 12–15 min until the hearts became softened and collapsed. The digested hearts were transferred to a dish, minced by scissors, and then dispersed to the cell suspension. The stop buffer (perfusion buffer containing 5 mg/mL bovine serum albumin and 0.1 mM CaCl₂) was added to the suspension to terminate the digestion. The isolated cells were filtered through a 100-μm strainer and centrifuged for 3 min at 50g to collect cardiomyocytes. The cardiomyocytes with different ploidy were identified by nuclear staining (NucBlue Live, Invitrogen, R37610) and isolated using pipettes.

MLO-Y4, MDA-MB-231, or RAW264.7 cells were seeded in 48-well plates (day 0) and treated with DMSO or Yoda1 (day 1). Cell seeding density differed between different durations of studies to avoid over confluence at the endpoint. After 2, 24, or 48 h incubation, cells were stained and incubated with 20 μL NucBlue^® Live (Ex: 360 nm, Em: 460 nm, all Invitrogen, Waltham, MA, USA) and 20 μL NucGreen^® Dead (Ex: 504 nm, Em: 523 nm, Invitrogen) stain for 30 min, following by capturing three random images per well using the fluorescence microscope (Nikon, Minato City, Tokyo, Japan). The images were analyzed using ImageJ to determine the number of cells. NucBlue^® Live stained all cells, while NucGreen^® Dead stained only dead cells. Positive control (PC) was prepared by treating cells with 70% ethyl alcohol for 15 min prior to staining.