Biorad automated cell counter

FET, HCT116 p21^−/−, HCT116, SW480, SW620 and Caco-2 cells were seeded into 96-well plate at a concentration of 20,000 cells with 100 μL/well DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic agent in a 37 °C incubator with 5% CO₂ and incubated for 24 hr. A range of concentrations (0, 1, 5, 10, 20, 40 μM) of PARPi were added to the seeded cells and incubated for 72 or 96 hr. Cell proliferation and cell survival were determined according to manufacturer’s instructions using either Cell Counting Kit-8 from Dojindo Laboratories (Rockville, MD) and/or the BioRad automated cell counter (Bio-Rad). The half maximal inhibitory concentrations (IC₅₀) of PARPi were determined by constructing a dose-response curve for each cell line. The experiment was performed at least three independent trials with at least three technical replicates.

MDA-MB-468 (sensitive) and MDA-MB-231 (resistant) cells were seeded at a density of 2.5 × 10⁵ cells in a 6-well plate in RPMI 1640 (1×) supplemented with 2.5% FBS to analyze the impact of cisplatin (Selleck Chemicals, Houston, TX, USA) and S63845 (Chemietek, Indianapolis, IN, USA) on growth and viability. Cells were placed into a humidified atmosphere of 5% CO₂ at 37 °C for 24 h before the medium was replaced and cells were dosed with various chemotoxic treatments. Technical triplicate cell counts were taken every 24 h throughout the 96 h timepoint for each treatment condition. Cells were collected through tryspin dissociation and washed with 1× dPBS. Equal parts of cell suspension and Trypan blue were mixed, and cell numbers and percent viability were recorded on a BioRad Automated Cell Counter (BioRad Laboratories, Hercules, CA, USA).

Cells were seeded 1 × 10⁶ in a 10 cm dish and treated through various conditions to modulate TAp73 and MCL1 expression. After 24 h of drug treatment, medium was removed and placed in a labeled 15 mL falcon tube. Tryspin dissociation was used to collect cells, and cells were washed with 1× dPBS and placed in corresponding tube. All cell counts were performed on a BioRad Automated Cell Counter (BioRad Laboratories, Hercules, CA, USA). Cells were pelleted for 5 min at 1000× g. Each FACs tube contained 5 μL propidium iodide (PI) staining solution (BD Pharmigen, San Diego, CA, USA, 51-66211E) and 5 μL FITC-Annexin V (BD Pharmigen, San Diego, CA, USA, 556419). A total of 5 × 10⁵ cells were resuspended in 1× Annexin V binding buffer (BD Pharmingen, 556454) and transferred to the corresponding FACs. Cells were incubated at RT in the dark for 15 min. After incubation, 450 μL of 1× Annexin V binding buffer was added, and FACS was collected on a BD LSRFortessa (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo (flowjo.com; version 10). Compensation controls used for this analysis included unstained cells, and cells stained for each individual fluorophore (Cy5-PI and FITC-Annexin V). Gating strategy eliminated cell debris and doublets through forward and side scatter plots.