Cell Viability, Necrosis, and Apoptosis Assays

Cell viability was determined colorimetrically with MTT. Briefly, cells were seeded in 96-well flat-bottomed plates at a density of 3 × 10⁴ cells per well in 200 μL of CM medium and incubated by 24 h at 37°C with 5% CO₂ as described above. Then, cell culture media were removed and replaced with serum-free DMEM. Following incubation in DMEM with FBS for 24 h, cells were incubated in the presence or absence of the indicated amounts of CDPs for 24 h at 37°C with 5% CO₂. To determine cell viability, MTT, 50 mg/mL in phosphate-buffered saline (PBS), was added to each well and incubated for 4 h at 37°C. Finally, 100 μL of 2-propanol/1 M HCl (19:1 vol/vol) was added to dissolve formazan crystals, and absorbance was measured at 595 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA).

To quantify necrosis and apoptosis, cell cultures were incubated with DMEM with FBS for 12 h prior to treatment with CDPs. Dimethyl sulfoxide (DMSO) was used as a control at the same concentration used to dissolve the CDPs. Following incubation, cells were collected by centrifugation at 2,000 g for 10 min. The pellet was suspended in 20 μL and incubated with annexin V and propidium iodide (PI) (Dead Cell Apoptosis Kit; Molecular Probes, Invitrogen Life Technologies, Carlsbad, CA, USA). Fluorescence was immediately quantified by fluorescence-activated cell sorting (FACS) using an Accuri-C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA). The percentages of fluorescent cells (PFC) and median fluorescence intensity were determined from histograms of the fluorescence emission in the plots, labeled as PFC or as relative fluorescence units. For apoptosis and necrosis assays, annexin V fluorescence was measured with fluorescence channel FL1 at 495/519 nm and for PI in FL2 channel at 535/617 nm. At least 20,000 cellular events were used for calculations.