Differentiation of NB4 cells following exposure to ATRA.
NB4 cells seeded at a density of 2 × 104/mL in RPMI 1640 media containing 10% FBS were left untreated (control) or treated with either all-trans retinoic acid (ATRA) at a concentration of 1 μM or vehicle (EtOH) for the indicated times. Differentiation was indicated as a decrease in the rate of proliferation, compared with controls, as measured by the direct counting method of viable cells (A); each data point represents the mean of 3 independent experiments with standard errors. NB4 differentiation was monitored by (B) a direct immunofluorescence assay using a phycoerythrin-conjugated monoclonal anti-CD11b antibody or isotype control. The percentage values of gated CD11b+ cells identified following 48 hours of ATRA or vehicle treatment were evaluated on the FL2 channel. This assay was carried out on all NB4 cells used in subsequent analyses; a representative output of 3 experiments is shown. Terminal differentiation of NB4 cells treated with EtOH (C) or ATRA (D) for 96 hours was analyzed by microscopic examination (original magnification × 1000) of glass slides following May-Grünwald Giemsa staining. Nuclear segmentation (arrow) and granule formation were observed in the ATRA-treated cells. A representative of 3 independent experiments is shown.
