Tryple express enzyme solution

RPMI-1640 medium was obtained from ATCC (Manassas, VA, USA). Fetal bovine serum (FBS), phosphate buffered saline (PBS, 0.01 M, pH 7.4), trypsin, penicillin-streptomycin (pen/strep) solution, crystal violet, dimethylsulfoxide (DMSO), cytochalasin-B and RNAse were obtained from Sigma-Aldrich (St. Louis, MO, USA). The TrypLE™ Express Enzyme solution was obtained from Gibco, Invitrogen (UK). The (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-Tetrazolium)-CellTiter 96^® AQueous One Solution Reagent (MTS) was obtained from Promega Corp. (Madison, WI, USA). The Giemsa dye, methanol, ethanol, acetic acid and propidium iodide (PI) were purchased from Merck (Darmstadt, Germany). The dihydrorhodamine 123 (DHR) and the Alexa Fluor^® 488 Annexin V/PI kit were acquired from Molecular Probes (Eugene, OR, USA). The 10 mM stock solution of DHR was prepared in DMSO, aliquoted, and stored under nitrogen at −20 °C. The MnTnHex-2-PyP⁵⁺ (MnP) was synthetized as described previously [20 (link)] (charges are omitted for clarity throughout the manuscript).

Plasmid pDsRed-MAX-N1 was purchased from Addgene (Watertown, MA, USA). DH5–α competent E. coli was purchased from New England Biolabs Inc (Ipswich, MA, USA). Plasmid Giga Kit was purchased from Qiagen (Germantown, MD, USA). Chitosan was purchased from Acmey Industrial (Shanghai, China). DAPI, Lipofectamine 3000, 2-Iminothiolane (Traut’s reagent), Succinimidyl Iodoacetate (SIA), Ultrapure Agarose, antibiotic-antimycotic, Tryple Express Enzyme solution, RPMI 1640 and DMEM cell culture medium were purchased from Invitrogen (Carlsbad, CA, USA). PD10 desalting columns, Sephacryl S-200 resin, and HyClone characterized fetal bovine serum (FBS) were purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA). NHS-Cy5 and NHS-AF488 were purchased from Lumiprobe Corp (Hunt Valley, MD, USA). Label IT Tracker Intracellular Nucleic Acid Localization Kits were purchased from Mirus Bio (Madison, WI, USA). SpectraPOR7 dialysis membrane was purchased from Repligen Corp (Waltham, MA, USA). All other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA).

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Chitosan (2.3 kMW) was obtained from Acmey Industrial (Shanghai, China). Branched PEI (MW 2 kDa) was purchased from Polysciences (Warrington, PA, USA). CleanCap EGFP mRNA was purchased from TriLink Biotechnologies (San Diego, CA, USA). Low molecular weight heparin used in PPH polymer was purchased from Galen Laboratory Supplies (North Haven, CT, USA). Furthermore, (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) (EDC), N-hydroxysuccinimide (NHS), Lab-Tek II 8-well chambered coverglass, NucBlue DAPI reagent, Lipofectamine 2000, LysoTracker Red DND-99, ultrapure agarose, antibiotic-antimycotic (100×), Tryple Express Enzyme solution, RPMI 1640, and DMEM cell culture medium were purchased from Invitrogen (Carlsbad, CA, USA). HyClone characterized fetal bovine serum (FBS) was purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA). Label IT Tracker Intracellular Nucleic Acid Labeling Kits were purchased from Mirus Bio (Madison, WI, USA). Cyanine5 NHS ester was purchased from Lumiprobe Corporation (Cockeysville, MD, USA). SpectraPOR7 dialysis membrane was purchased from Repligen Corp (Waltham, MA, USA). Moreover, 4T1, HepG2 and C6 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA).

Cells were lifted using TrypLE Express Enzyme solution (Life Technologies, Carlsbad, CA), fixed in BD Cytofix buffer (BD Biosciences, San Jose, CA) for 20 min at room temperature and permeabilized with BD Permeabilization/Wash buffer (BD Biosciences, San Jose, CA) at 1 × 10⁷ cells per 1 ml for 10 min. Cells were stained with the following primary antibodies: mouse anti‐human Sox9 (Abcam, Cambridge, MA; 76997, 1:200), mouse anti‐human CD44‐PE/Cy7 (Abcam 46793, 1:100), mouse anti‐human CD90‐FITC and mouse CD105‐Alexa 647 (both from R&D, Minneapolis, MN; FMC002), for 30 min at the concentration suggested by the manufacturer. Secondary antibody (donkey anti‐mouse IgG Alexa 488, Invitrogen, Carlsband, CA) was diluted 1:250. Cells were scanned using a LSR II flow cytometer and analyzed with Flowjo software.

Human iPSC line hCBiPSC2 was derived from the human cord blood endothelial cells, as described before [72 (link)]. In brief, iPSCs were cultured under xeno-free and feeder-free conditions. In brief, 12 well non-tissue culture-treated plates (Falcon™, Corning, Corning, NY, USA) were coated with human recombinant Laminin-521 (BioLamina, Sundbyberg, Sweden), followed by seeding of the cells in 50,000 cells/cm² density and culturing under sterile conditions in StemMACS medium (Miltenyi Biotec, Bergisch Gladbach, Germany) at 5% CO₂ and 37 °C in humidified CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA) with a daily medium change. Harvesting of the cells was performed with application of TrypLE™ Express Enzyme solution (Life Technologies, Carlsbad, CA, USA).

Wild-type SH-SY5Y (neuroblastoma) cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (Gibco), supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin, and grown in a humidified atmosphere of 5% CO₂, at 37°C. They were seeded into 35mm glass bottom dishes using TrypLE express enzyme solution (Gibco) at a density of 10⁶ cells per dish in 3mL of complete media. After 24 hours, the cells were washed twice with PBS and fixed using 4% PFA for 15 minutes at room temperature (RT), after which they were washed three time with PBS and labeled using a Wheat Germ Agglutinin conjugate of Alexa Fluor^TM 555 (Thermo Fisher Scientific). The label was applied to cells at a concentration of 5 μg/mL in PBS and incubated for 10 minutes at RT. Cells were then washed three times in PBS and stored in PBS at 4°C until needed for imaging.
Immediately before imaging, the cells were drained, then a slice of a cleared brain tissue placed on top of the cell layer. Residual TDE was removed with a lens cleaning tissue, then the slice was attached punctually on its perimeter with a few drops of cyanoacrylate (“superglue”) and left to dry for 10 minutes. After this, the dish was filled with 100% TDE to cover the tissue, and agitated manually for 5 minutes to remove any RI gradients, as assessed by a naked eye.