Acridine orange

SCP1 cells and primary chondrocytes were transfected with synthetic Link N mRNA (1 µg/1 × 10⁵ cells). After 24 h, the cells were detached, loaded into a Via1-Cassete™, which is coated with Acridine Orange and DAPI (ChemoMetec, Kaiserslautern, Germany). Using the NucleoCounter^® NC-200™, the cell count and viability were determined. All cells stained with Acridine Orange appeared green and non-viable cells stained with DAPI appeared blue. Cell viability was calculated using the NucleoCounter software.

The suspension-adapted Trichoplusia ni BTI-TN-5B1-4 cell line (High Five, cat. num. B85502, Thermo Fisher Scientific, Grand Island, NY, USA) was grown in the low-hydrolysate animal origin-free Sf900III medium (Thermo Fisher Scientific). Cells were subcultured three times a week at a density of 2–4 × 10⁵ cells/mL in 125 mL disposable polycarbonate Erlenmeyer flasks (Corning, Steuben, NY, USA), as previously described [16 (link)]. All cultures were grown in an orbital shaker at 130 rpm (Stuart, Stone, UK) and maintained at 27 °C. Cell count and viability were measured with the Nucleocounter NC-3000 (Chemometec, Allerød, Denmark) using acridine orange for cell detection and 4′,6-diamidino-2-phenylindole (DAPI) (Chemometec) to quantify non-viable cells.

3,4-dihydroxy-L-phenylalanine (L-DOPA), synthetic melanin, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), Dulbecco’s phosphate-buffered saline (DPBS) with CaCl₂ and MgCl₂, penicillin G, amphotericin B, SIGMAFAST™ Protease Inhibitor Coctail Tablet, and Phosphatase Inhibitor Coctail 3 were purchased from Sigma Aldrich Inc. (St. Louis, MO, USA). Neomycin sulfate was obtained from Amara (Gdynia, Poland). Medium M-254, human melanocyte growth supplement-2 (HMGS-2), and trypsin/EDTA solution were obtained from Cascade Biologics/Gibco (Carlsbad, CA, USA). Solution 5 (400 µg/mL VitaBright-48™, 500 µg/mL propidium iodide, and 1.2 µg/mL acridine orange in DMSO) was obtained from ChemoMetec (Allerod, Denmark). Cell Proliferation Reagent WST-1 was purchased from Roche (Mannheim, Germany). The remaining chemicals were purchased from POCH S.A. (Gliwice, Poland) or Sigma Aldrich. UV-visible spectra were recorded on a Jenway 7315 Spectrophotometer (Staffordshire, UK) and HITACHI Fluorescence Spectrophotometer F-7000 (Hitachi High-Tech, Tokyo, Japan).

Image cytometry with an NC-3000 Image Cytometer (ChemoMetec, Allerod, Denmark) was used for cellular and mitochondrial phenotyping. A minimum number of 5000 events were analyzed in each experiment. The following assays were executed according to the manufacturer’s recommendations: cell count (acridine orange and DAPI (ChemoMetec)), mitochondrial membrane potential (JC-1 and DAPI (ChemoMetec)), thiol redox status (VitaBright-48, Propidium iodide, and acridine orange (ChemoMetec)). Mitochondrial superoxide levels (MitoSOX™ (Thermo Scientific) and Hoechst-33342 (ChemoMetec)) were measured as described earlier [43 (link)]. To measure mitochondrial mass, 100 nM fluorescent dye MitoTracker™ Green FM (Invitrogen, Carlsbad, CA, USA) diluted in Hank’s balanced salt solution (HBSS) (Invitrogen) was added to cells for incubation for 30 min at 37 °C. After removing excess dye, the cells were collected and a 1:1000 dilution of RedDot2 (Biotium, Fremont, CA, USA) (concentration not specified) was added to each sample before measurement. The NucleoView NC-3000 software (ChemoMetec) was used for data analysis.

hESC lines at early passages were harvested in a batch of five to 10 vials as seed stocks with 5 × 10⁵ to 2 × 10⁶ viable cells per vial. To prepare master cell stocks, we thawed one vial from seed stocks, expanded the cells in culture, and then banked the cells in batches of 50 vials with >1 × 10⁶ viable cells per vial in cryopreservation medium consisting of StemFit AK03N with 10% DMSO (Sigma–Aldrich, St. Louis, MO, USA) and Y-27632 (final concentration; 5 μM). The number of viable collected cells was determined by double staining with acridine orange and DAPI (ChemoMetec). The vials were labeled for traceability and placed in a CoolCell freezing container (Biocision, San Rafael, CA, USA) in a −80 °C mechanical freezer (PHC, Tokyo, Japan). The next day, the vials were transferred to the vapor phase of a liquid nitrogen tank (LN2 supply tank, Taiyo Nippon Sanso, Tokyo, Japan).

Cell viability was assessed using the NucleoCounter^® NC-3000™ fluorescence image cytometer (ChemoMetec, Lillerød, Denmark) according to the Cell Viability and Cell Count Assays protocol. In brief, after the trypsinization, the cells were centrifuged and suspended in the growth medium and then loaded into the Via1-Cassette™ containing two fluorescent dyes: DAPI and acridine orange (ChemoMetec, Lillerød, Denmark). The immediate analysis causes DAPI penetration only through a damaged and permeable cell membrane of dead cells. In turn, acridine orange allows the detection of the total cell population.