Supernatants of OP9 stromal cells cultured with supernatants of p66Shc-deficient leukemic Eμ-TCL1 cells enhance B-cell adhesion and migration. (A) Scheme of the experimental strategy for the pseudo-emperipolesis and Transwell assays. (B) Migration of R1-gated live splenic CD19⁺ cells from WT (n = 3) mice measured after a 3-hour treatment with supernatants of OP9 stromal cells kept either in culture medium or conditioned for 48 hours with the culture supernatants (SN) from WT or p66Shc^−/− (p66^−/−) cells or leukemic B cells purified from Eμ-TCL1 (TCL1) or Eμ-TCL1/p66Shc^−/− (TCL1/p66^−/−) mice with overt disease, washed, and cultured in fresh medium for an additional 48 hours. The data, obtained from triplicate samples from each mouse, are presented as mean migration index (ratio of migrated cells in chemokine-treated/untreated samples) ± standard deviation. (C) Quantification by flow cytometry of the percentage of splenic CD19⁺ cells (gated on live lymphocytes) from WT (n = 3) mice that remained adherent to OP9 stromal cells (pseudo-emperipolesis assay) grown on 48-well plates in culture medium or conditioned for 48 hours with the supernatants from WT B cells (n = 3) or p66Shc^−/− B cells (p66^−/−) or leukemic B cells purified from Eμ-TCL1 mice (TCL1; n = 3) or Eμ-TCL1/p66Shc^−/− (TCL1/p66^−/−; n = 3) mice with overt disease, washed, and cultured in fresh medium for an additional 48 hours. Before counting, splenocytes were labeled with anti–CD19-FITC antibodies. The data, which refer to quadruplicate samples from 3 independent experiments, are presented as the percentage of total input cells that remained attached to each well. Migration (D) and adhesion (E) of leukemic Eμ-TCL1 cells (TCL1; n = 3) or Eμ-TCL1/p66Shc^−/− cells (TCL1/p66^−/−; n = 3), stained with DiO and DiL fluorescent dyes, respectively, and mixed prior to the assays. Data in bar graphs are mean ± standard deviation. ***P ≤ .001, **P ≤ .01, *P ≤ .05, 1-way ANOVA, multiple comparisons. FSC, forward scatter; SSC, side scatter; SN, supernatant.