Assessing TIGK Cell Metabolic Activity

The metabolic activity of TIGK cells was assessed by measuring total ATP levels using the CellTiter-Glo reagent (Promega, Madison WI), as described by the manufacturer. TIGK cells were seeded at a density of 6 × 10⁴ cells in 1 mL media per well and incubated at 37°C, 5% CO₂ for 24 h in a 12-well flat bottom plate. Cells were then incubated with BAR or 10:90 PLGA-PEO BAR-EFs (1.3 or 3.4 μM) for 24 h at 37°C in 5% CO₂. Cells were then lysed with 500 μL of 0.1% Triton X-100 for 30 min at 37°C. The lysates were collected and centrifuged at 1,000 × g for 10 min at 4°C, and 50 μL of supernatant was mixed with 50 μL of CellTiter-Glo reagent. Samples were incubated at ambient temperature for 10 min in a black 96-well plate in the dark. Total luminescence was measured with a Victor 3 luminometer (Perkin-Elmer, Inc.). Cells incubated with 1 ng of staurosporine or with medium-only served as positive and negative controls for cell death, respectively (Mahmoud et al., 2019 (link)).