2.7. Cell Cycle Analysis by Flow Cytometry

The cells were seeded at a density of 20 × 10⁴ cells/mL in a 35 mm petri dish containing 3 mL of complete media. The cells were incubated at 37 °C and 5% CO₂ until 90% confluence and the media was replaced every 2 days. The cells were treated with either free PTX, free LAP, PTX NPs, LAP NPs at the IC-50 concentration, or left untreated for 48 h at 37 °C. After 48 h treatment, the cells were stained with Propidium Iodide (PI Flow Cytometry Kit, Abcam, Cambridge, MA, USA) for flow cytometry according to the manufacturer’s instructions. Briefly, the cells were trypsinized and the aspirated medium and PBS were collected to minimize cell loss. The cells were centrifuged at 700× g for 5 min as necessary. The cells were washed with 1X PBS and fixed with 66% ethanol by slowly adding ethanol to PBS during vortexing. The cells were stored in ethanol at 4 °C for at least 2 h and up to 4 days. The cells were centrifuged and washed with PBS to remove the ethanol. The 1× Propidium Iodide and RNase solution was prepared immediately prior to use by mixing 5% v/v of 20× Propidium Iodide and 0.05% v/v 200X RNase in 1X PBS. Then the cells were resuspended in 200 µL/500,000 cells of 1X Propidium Iodide and RNase solution and incubated in the dark at 37 °C for 30 min. Prior to flow cytometry, the cell samples were stored on ice and filtered through a cell strainer (Falcon Test Tube with Snap Cap, Fisher Scientific, Pittsburg, PA, USA). Flow cytometry was performed on a BD FACSCanto™ II Analyzer (BD Biosciences, San Diego, CA, USA) and 10,000 cells were analyzed at an excitation of 488 nm and emission of 670 nm. The samples were analyzed in triplicate.