Effects of Notch ligand-dependent activation on HL-60 differentiation. HL-60 cells were washed and seeded on NIH3T3pBABE and NIH3T3pBABE-J2 cell monolayers at the density of 0.2 × 106/mL when in the absence and at the density of 0.4 × 106/mL when in the presence of ATRA or TPA. At days 2, 3, and 4 of coculture, HL-60 cells were detached from the 3T3 layer, washed, and labeled with anti-CD11b PE-conjugated and anti-CD14 FITC-conjugated MoAbs and subsequently analyzed by two-color flow cytometric analysis. (A) Two-color flow cytometric analysis at day 4 of differentiation in the absence and in the presence of ATRA or TPA. Contour plots represent fluorescence intensity for CD11b on the x-axis and for CD14 on the y-axis. Contaminating NIH3T3 cells were excluded from the analysis based on their different forward scatter (FSC) and side scatter (SSC) and their negativity to CD15 (positive 100% on HL-60 cells). (B) Values in the line graphic represent the mean percentage of HL-60 cells expressing CD11b during coculture with NIH3T3pBABE and NIH3T3pBABE-J2 at days 0, 2, 3, and 4 after induction of differentiation. Bars represent standard error.
