Presence of IL3 during the adhesion assay.

Presence of IL3 during the adhesion assay prevents up-regulation of p27^KIP1 and allows up-regulation of cyclin-E. Mobilized blood CD34⁺ cells cultured for 48 hours in serum-free medium with low-dose cytokines were incubated with 8A2 and plated, with or without 10 ng/mL IL3, in PLL- or FN-coated wells for 12 to 16 hours. Adherent and nonadherent cells were collected separately, fixed, permeabilized, washed, and incubated with antibodies directed at p27^KIP1 and cyclin-E (open histogram) or isotype control (closed histogram) at room temperature for 30 minutes, followed by FITC-conjugated goat antimouse immunoglobulin. A representative example of 3 individual experiments is shown. Adhesion to PLL in the presence or absence of IL3 did not affect the expression level of cyclin-E or p27^KIP1 (not shown). However, up-regulation of p27^KIP1 and down-regulation of cyclin-E levels are prevented when IL3 is present during the adhesion assay.