Propidium iodide dna staining kit

MDA-MB-231 (Caucasian American) and MDA-MB-468 (African American) TNBC cells were purchased from the American Type Culture Collection (ATCC). Fetal bovine serum heat-inactivated (FBS-HI), Dulbecco’s modified Eagle’s medium (DMEM) high glucose, penicillin/streptomycin, and phosphate-buffered saline (PBS) were obtained from Genesee Scientific (San Diego, CA, USA). Alamar Blue^®, fucoxanthin (95% purity), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Human Angiogenesis Antibody Array (Cat# AAH-ANG-1000-4) and TNF-alpha (Cat# ELH-TNFα) were purchased from RayBiotech (Norcross, GA, USA). PCR arrays, SYBR Green, and iScript advanced reverse transcriptase kit were purchased from Bio-Rad (Hercules, CA, USA), and the propidium iodide DNA staining kit was purchased from Abcam (Boston, MA, USA). Wes kit and reagents were attained from ProteinSimple (San Jose, CA, USA), and antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) and ThermoFisher (Waltham, MA, USA).

Flow cytometry analysis using a propidium iodide DNA staining kit (Abcam USA) was employed to assess the impact of fucoxanthin on cell cycle progression in MDA-MB-231 and MDA-MB-468 cells. Cells were seeded at 1 × 10⁶ cells/flask and incubated overnight. The cells were treated with various concentrations of fucoxanthin (1.56–12.5 µM) and incubated for another 24 h. Meanwhile, the control cells were treated with DMSO at a concentration of <0.1%. Cells from each flask were harvested, pelleted, and washed with cold PBS. Cells were fixed in cold HPLC ethanol and vortexed; all clumps were removed. Before analysis, cells were centrifuged, and ethanol/PBS was aspirated. Cells were then treated with 250 μL of a cocktail (PBS, PI, and ribonuclease) and incubated for 30 min. Lastly, cell cycle phases were quantified using the FACSCalibur Flow cytometer (Becton Dickinson, San Jose, CA, USA). Each sample of 1 × 10⁴ cells was examined and analyzed using the CELLQuest software Pro/Version 5.1 (BD Biosciences, San Jose, CA, USA).