HL-60 Leukemia Cell Differentiation

The HL-60 human myeloblastic leukemia cells were originally an early pass of cells derived from the original patient samples, generously gifted by Dr. Robert Gallagher and continuously maintained in this laboratory as previously published [23 (link)]. The cells used were certified as mycoplasma free HL-60 by Bio-Synthesis, Lewisville, TX, in August 2017. The HL-60 cells were grown in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT) and 1x antibiotic/antimycotic (Sigma, St. Louis, MO) in a 5% CO2 humidified atmosphere at 37°C. Cells were continuously cultured in the constant exponential growth phase, not exceeding 2.0 x10⁶, with cell viability exceeding 95% by 0.2% trypan blue (Invitrogen, Carlsbad, CA) exclusion dye via hemocytometry. Experimental cultures were initiated at a density of either 0.1 x 10⁶ for 24, 48, and 72 hour time points for flow cytometry, or 0.2 x 10⁶ for 8 hour flow cytometry time points and 48 hour lysate collection. All cells and lysate collected used the same treatment conditions of 0 μM or 10 μM RRD-251 Hydrochloride (Sigma) and 0 μM or 0.5 μM 1,25-dihydroxyvitamin D3 (Cayman, Ann Arbor, MI). Cells were harvested at 8, 24, 48, and 72 hours post-treatment. Three biological replicates of each experiment were performed unless stated otherwise.