Trypan blue

The cell viability of the 661w photoreceptor-like cell line was determined by a Trypan blue assay using an Countess^TM II FL automated cell counter (Life Technologies Corporation, Bothell, WA, USA). After harvesting the cells, 15 μL of cell suspension was mixed with 15 μL of Trypan blue (T10282, Invitrogen) and rested for 30 s before injection to the automated cell counters for analysis.

Cells in logarithmic growth phase were seeded in 6-well plates at a density of 100,000 cells per well and treated with different concentrations of MTX (0-1 μM). Cells were harvested after 24, 48, and 72 hours and counted under a light microscope after trypan blue exclusion (0.4% trypan blue, Life Technologies, USA) according to instructions of manufacturer. Each treatment was performed in triplicate wells per experiment.

The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) assay was used to assess cellular viability. For anti-cancer treatments, cells were treated with 5-Fluorouracil (5-FU; Sigma-Aldrich, St. Louis, MO, USA), with concentrations ranging from 5.0 × 10⁻⁴ to 7.6 × 10⁻⁹ M for 72 h. The cells were assessed for their viability by adding 0.5 µg/mL MTT per well. Insoluble formazan crystals were dissolved by adding an acid-isopropanol stop solution (0.04 N HCl). Absorbance was measured at 610 nm, using the Tecan-Sunrise microplate reader (Tecan, Männedorf, Switzerland). For trypan blue count, cellular samples were mixed 1:1 with 0.4% trypan blue (Thermo Fisher Scientific, Waltham, MA, USA). Cells permeable to trypan blue were counted as dead. Counts were performed in triplicate by using a Bürker chamber and the Eclipse Ts2 inverted microscope (Nikon, Melville, NY, USA).

To discriminate between binding and internalization of GFP-BCG, we carried out reduction in green fluorescence of Trypan blue quenched bacteria by excitation energy transfer [26 (link),27 (link)]. To this end, we washed infected or uninfected bone-marrow-derived macrophages in cold phosphate buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2.0 mM KH₂PO₄; pH adjusted to 7.4) and incubated them in PBS on ice for 15 min. Cells were harvested, normalized to a concentration of 1 × 10⁶ cells/50 µL in PBS, and either fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich, Steinheim, Germany) for 10 min at room temperature or processed to quench adherent bacteria. To quench the fluorescence of adherent GFP-BCG, 500 µL Trypan blue (0.4% w/v in PBS; Corning Inc., Corning, NY, USA) was added for 1 min, followed by washing twice in PBS and fixation in 4% PFA for 10 min at room temperature. Quenching with Trypan blue reduced the GFP fluorescence of only adherent bacteria (not internalized bacteria) by excitation energy transfer [26 (link),27 (link)]. Bacterial binding (without Trypan blue treatment) and internalization (with Trypan blue treatment) were analyzed by Attune NxT flow cytometry (Thermo Fisher Scientific, Waltham, MA, USA) and FlowJo software v10 (FlowJo LLC, Ashland, OR, USA).

Trypan blue (Thermo Fisher, Waltham) staining was used to evaluate the cell viability. One milliliter of resuspended ADSCs was mixed with an equal volume of Trypan blue, and the viability was assessed using an Invitrogen™ Countess™ II FL (Thermo Fisher, Waltham, USA).

Edited hESCs were cultured in E8 medium for 2–4 days in a 24‐well plate when cells cover approximately 80% of the surface area of the culture vessel and then exposed to 10 nM AP1903 (MCE, HY‐16046) for the desired amount of time. The edited hESCs treated with AP1903 were harvested by TrypLE digestion followed by staining with Trypan blue (Gibco, 15250061) and annexin V (AnV; Abcam, ab14147) according to the manufacturer's instructions. Live and dead cells stained with Trypan blue were counted by Countess II (Thermo Fisher Scientific). Cells stained with AnV were quantified and analysed by FCM (BD LSRFortessa) and FlowJo 10.5.3. Cell morphology and number change were also assessed by microscopy at representative time points.